Humanized antibodies against human interferon-alpha

ABSTRACT

The present invention provides humanized anti-human IFN-α monoclonal antibodies useful for therapeutic applications in humans. Preferred antibodies are humanized versions of murine antibodies ACO-1 and ACO-2, as well as variants thereof.

This application is a continuation of U.S. application Ser. No.12/597,357, filed Mar. 17, 2010 now U.S. Pat. No. 8,163,885, which was a35 U.S.C. §371 national stage application of International PatentApplication PCT/EP2009/055448, filed May. 6, 2009, which claimedpriority of European Patent Application EP 08103847.3, filed May 7,2008, this application further claims priority under 35 U.S.C. §119 ofU.S. Provisional Application No. 61/056,193, filed May 27, 2008, theentire contents of each of which are hereby incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to humanized antibodies against humaninterferon alpha (IFN-α) and their use in treating or preventing variousdiseases and disorders in human patients.

BACKGROUND OF THE INVENTION

Based on a variety of different observations, interferon alpha (IFN-α)is a cytokine believed to be involved in a number of autoimmunediseases. Although systemic lupus erythomatosus (SLE) patients often donot have measurable serum levels of IFN-α, they appear to have a clearIFN-α gene signature. In addition, induction of dendritic cell (DC)maturation by treatment of DCs with SLE patient serum can be inhibitedby an anti-IFN-α antibody. It has also been shown that knockout of theIFN-α/β receptor in New Zealand Black (NZB) mice having an SLE phenotyperesults in a near normal phenotype (Santiago-Raber et al., J Exp Med.2003; 197(6):777-88).

Antibodies against IFN-α have therefore been suggested as tools toneutralize the activity of this cytokine for the treatment of suchautoimmune diseases, alone or in combination. Specific murine antibodies(ACO-1 to ACO-6) that recognize a wide range of different IFN-α subtypeswere generated and characterized as described in the internationalpatent application published as WO20060086586. However, murineantibodies are not suitable for use in humans because of theirimmunogenicity, and it is therefore desirable to generate humanizedantibodies where the murine CDRs are grafted onto a human scaffoldantibody. However humanized antibodies often suffer from functionaldeficiencies as compared to the murine parent, such as, e.g., a loweraffinity and/or stability and/or undesirable immunogenicity. Suchdeficiencies in humanized antibodies can in some cases be compensatedfor by making one or a few back point mutations. It is usually desirableto perform no or only a very few back point mutations since the presenceof too many back mutations tend to result in undesirable low stabilityand/or an undesirable degree of immunogenicity. The provision of a safeand stable humanized anti-IFN-α antibody having desirable biologicalproperties such as e.g. retaining affinity and potency of the humanizedanti-IFN-α antibody to a large number of IFN-α subtypes is thusdesirable.

There is thus a need in the art for humanized anti-IFN-α antibodieshaving desirable features with respect to features such as, e.g.,stability, specificity, safety, immunogenicity, etc. Furthermore, thereis a need in the art for efficient methods for producing suchantibodies.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a humanized antibodythat specifically binds human interferon-α (IFN-α), or anantigen-binding fragment thereof, which humanized antibody is ahumanized version of murine antibody ACO-1 or ACO-2, or of a combinationthereof, comprising fewer donor amino acid residues than the murinecomplementary determining regions (CDRs) according to Kabat.

In another aspect, the present invention furthermore relates to ahumanized antibody that specifically binds IFN-α, or an antigen-bindingfragment thereof, wherein said antibody is capable of binding IFN-αsubtypes A, 2, B2, C, F, G, H2, I, J1, K, 4a, 4b and WA, but notsubtypes 1 or D, and wherein said antibody comprises fewer donor aminoacid residues than the non-human CDRs according to Kabat.

The present invention furthermore relates to methods for obtaining suchantibodies as well as use of such antibodies for therapeutic purposesand compositions comprising such antibodies.

The antibodies according to the present invention can be suitable fortreatment of various inflammatory diseases.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows analysis of murine ACO-1 VH (A) and VL (B) sequences forhumanization (hz=humanized), where the mask is shown in shaded text,Kabat CDRs are shown in bold, differences between the mouse sequence andthe human germline sequence are shown in underlined text, potentialsomatic hypermutated residues are shown in bold underlined text, andpotential back-mutation residues in shaded underlined text. ACO-1 VH=SEQID NO:1; human germline VH1_(—)46/JH4=SEQ ID NO:2; hzACO-1 VH=SEQ IDNO:3; ACO-1 VL=SEQ ID NO:4; human germline VKIII_L6/JK2=SEQ ID NO:5;hzACO-1 VL=SEQ ID NO:6.

FIG. 2 shows an alignment between ACO-1 and ACO-2 VH (A) and VL (B)sequences, as well as the corresponding mouse germline sequences. ACO-2VH=SEQ ID NO:7; mouse germline J558.33/D_/JH3_(—)1=SEQ ID NO:8; ACO-2VL=SEQ ID NO:9; mouse germline ae4/JK4_(—)1=SEQ ID NO:10.

FIG. 3 shows the location of ACO-2 residues selected for introductioninto hzACO-1.

FIG. 4 shows hzACO-1 inhibition of the protective effect of allinterferon subtypes tested except for IFN-αD and IFN-α1 in a CPE assay.

FIG. 5 shows hzACO-1 inhibition of 12 IFN-α species by a reporter gene(RG) assay. Values for each antibody concentration were normalized andthe average of four repetitions were calculated. Data are shown asaverage+/−standard errors. Best fit sigmoidal response curves werecalculated using Prism software. R2 values were for all dataset above0.98 (except for IFN-αD for which no curve fitting was made).

FIG. 6 shows an RG assay comparison of hzACO-1 with a hzACO-1 variantshaving the single ACO-2-derived mutation A93V, using IFN-αA (A) orIFN-αF (B). Data calculations were made as described for FIG. 5.

FIG. 7 shows transition temperatures for hzACO-1 and variants at pH 3.5(A), 4.5 (B), and 5.5 (C), without additives.

FIG. 8 shows the structure of hzACO-1 Fab fragment chains (H, L) boundto IFN-α8, determined by X-ray crystallography.

FIG. 9 shows the IFN-α8 binding epitopes for IFNAR1 and IFNAR2(Quadt-Akabayov S. R. et al. Protein Sci. 15, 2656-2668, 2006 andRoisman L. O et al. J. Mol. Biol. 353, 271-281, 2005), as indicated bycolored boxes below the IFN-α8 sequence. Residues indicated by boxescolored gray are partly conserved among all IFN-α subtypes whileresidues indicated by black colored boxes are fully conserved. ThehzACO-1 binding epitope, using a 4 Å distance cut-off, on IFN-α8 isindicated by “*” above the amino acid sequence of IFN-α8.

FIG. 10 shows comparison of the mouse ACO-1 mAb to hzACO-1 as well astwo variants hereof in the RG assay. One variant is a humanized ACO-1harboring the entire CDRH2 (designated hzACO-1-kabat CDRH2) whereas thehzACO-1 was constructed with a shorter CDRH2 as described in example 2.in addition the figure shows another mutated hzACO-1 which has beenoptimized for interaction with IFN-αs (hzACO-1 Y32E, T30R) throughrational design. These four recombinant mAb variants were compared withrespect to inhibition of five different representative IFN-α subtypes,as indicated.

FIG. 11 shows a protein stability study of hzACO-1 expressed with humanIgG1, IgG2 and IgG4 isotypes. Aggregation was determined by HPLC afterincubation in histidine buffer for 5 weeks.

FIG. 12 shows a ⁵¹Cr release assay illustrating lack of ADCC by hzACO-1IgG4, IFN-α and different combinations thereof, at differenteffector:target cell ratios (E:T). Cells+PBMCs alone without IFN-α prhzACO-1 determines background lysis and Triton®-X 100 detergentillustrates maximal lysis. Rituxan® was included as a positive controland induces detectable cell lysis at all E:T ratios.

FIG. 13 shows a complement binding study by Elisa. The hzACO-1 expressedas an IgG4 was unable to fix complement when bound to IFN-α. As apositive control the hzACO-1 was cross bound with an anti-IgG4 pAb, anda clear dose dependent binding of C4 to the anti-IgG4 was detected.

DESCRIPTION OF THE INVENTION

The present invention is based, in part, on anti-IFN-α antibodies withproperties suitable for treating human patients suffering from anIFN-α-related condition or disease, such as, e.g., a lupus disease ordisorder such as, e.g., SLE; graft versus host disease; type 1 diabetes,AIDS, autoimmune thyroiditis, psoriasis, juvenile dermatomyositis, andSjögren's syndrome. The antibodies are typically based on humanizedversions of the murine ACO-1 and/or ACO-2 antibodies.

ACO-1 and ACO-2 were identified as capable of blocking the bioactivityof thirteen recombinant IFN-α subtypes as well as two complex mixturesof IFN— produced upon viral infection (see WO2006086586). ACO-1 andACO-2 also consistently blocked the bioactivity of serum from SLEpatients that exhibited IFN-α signatures by microarray analysis. ACO-1and ACO-2 did not significantly neutralize the bioactivity of IFN-αprotein subytpes D and 1, but did neutralize the IFN-α bioactivity ofSLE serum. Though not limited to theory, it is therefore possible thatsubtypes D and 1 are not significantly involved in the etiology of SLE.

As described in the Examples, structural modelling of the variableregions revealed that it was possible to humanize ACO-1 and ACO-2 usingfewer donor (murine) residues than the Kabat CDRs, thus further reducingthe risk for an adverse immune response in a human patient. The analysisalso identified advantageous sites for back-mutations. It was furtherdiscovered that, possibly due to the high sequence similarity betweenthe ACO-1 and ACO-2 variable regions (differing only at 13 sites),certain amino acid residues in the humanized ACO-1 (hzACO-1) sequencecould be replaced by ACO-2 residues at the corresponding position.Within CDR regions, mutations can normally be made without rendering theantibody sequence less human. This humanization procedure resulted inimproved functional properties such as affinity, stability, expressionlevel and IFN-α-inhibitory activity of the humanized antibody.

In a humanized ACO-1 antibody, exemplary mutations in the hzACO-1 VH(SEQ ID NO:3) include V5Q, T28S, M69L, R71V, T73K, S761, S76N, T771,V78A, Y79F and A93V, as well as any combination thereof, using Kabatnumbering. Exemplary mutations in the hzACO-1 VL (SEQ ID NO:6) includeE1Q, D29G, L33F, L47W, S50G, I58V, and F71Y, as well as any combinationthereof. In one embodiment, the hzACO-1 VH region comprises a mutationselected from T28S, N31S, and A93V. In another embodiment, the hzACO-1VH region comprises a mutation selected from T28S, N31S, and A93V, andany combination thereof, such as, e.g., T28S and N31S, T28S and A93V,and N31S and A93V. In another embodiment, the hzACO-1 VH regioncomprises a mutation selected from T28S, N31S, and A93V, or acombination thereof, such as, e.g., T28S and N31S, T28S and A93V, orN31S and A93V; and at least one additional mutation.

Definitions

To facilitate the understanding of this invention, a number of terms aredefined below.

“ACO-1 and ACO-2 antibodies” are characterized and described inWO20060086586. ACO-1 is deposited with ATCC accession no. PTA-6557(WO2006086586) and ACO-2 is deposited as ATCC accession No. PTA-7778(WO2008021976). The antibodies according to the present invention arehumanized variants of ACO-1 and ACO-2. However, the humanized versionsof ACO-1 and ACO-2 according to the present invention do not comprisethe full length murine Kabat sequences. In a preferred embodiment, atleast one of the CDR sequences comprise a truncation of about 3-10 aminoacids, preferably 3-8, more preferably 4-7 amino acids. The antibodypreferably comprises a truncation in the CDR H2, said CDR H2 preferablybeing truncated by 3-10, preferably 3-8, more preferably 4-7, and mostpreferably by 6 amino acids. And it furthermore follows that occasionalpoint mutations may be introduced in one or more CDR sequences as wellas within the human scaffold antibody. The term “ACO-1 and ACO-2antibodies” may however furthermore embrace any IFN-α antibody capableof binding IFN-α subtypes A, 2, B2, C, F, G, H2, I, J1, K, 4a, 4b andWA, but not subtypes 1 or D. It should however be understood that ACO-1and ACO-2 as such may be perceived as only one antibody since thedifferences in their CDR sequences are only a few amino acids. It isplausible that ACO-1 and ACO-2 thus represent two stages of in vivosomatic hypermutation of the same antibody. An ACO-1/ACO-2 antibodyaccording to the present invention is thus a humanized antibodycomprising CDR sequences having at least 90% identity with the CDRsequences of ACO-1 and ACO-2, more preferably at least 92%, and mostpreferably at least 95%.

The terms: “CDR truncation”, “forward mutation”, and “shortening ofCDRs” may be used interchangeably throughout the document. In connectionwith the present invention such terms generally refer to the fact thatCDR-truncation may be perceived as a number of forward mutations in arow—meaning that a shortened murine CDR fragment can be grafted onto thehuman framework. While it may not be surprising that grafting of shorterCDRs tend to result in antibodies with reduced degree of immunogenicity,it is actually surprising that other advantageous features of thehumanized antibody may be retained such as e.g. stability, specificity,etc. “Back mutations” always refer to mutations in the framework (i.e.not in the CDRs)—and back mutations are typically introduction of one ormore “murine” amino acid residue at selected sites e.g. in order tostabilise the antibody structure.

The term “interferon alpha” (IFN-α), as used herein, refers to a familyof proteins that include some of the main effectors of innate immunity.There are at least 15 known subtypes of human IFN-α. The names of theIFN-α protein subtypes and corresponding encoding genes are listed inTable 1.

TABLE 1 IFN-α protein subtypes and genes IFN-α protein subtypeCorresponding IFN-α gene A  2a 2  2b B2  8 C 10 D (Val¹¹⁴)  1 F 21 G  5H2 14 I 17 J1  7 K  6 4a  4a 4b  4b WA 16 1 (Ala¹¹⁴)  1

See Pestka et al. (1997) “Interferon Standardization and Designations” JInterferon Cytokine Res 17: Supplement 1, S9-S14. IFN-αB2 is sometimesalso referred to as IFN-αB, and is not to be confused with IFN-β.Natural IFN-α from leukocytes (leukocyte IFN-), as well as recombinanthuman IFN-α protein subtypes are available from PBL Biomedical Labs,Piscataway, N.J. (interferonsource.com). Natural IFN-α is a complexmixture of IFN-α subtypes. Methods for detecting and quantifying theseinterferons, such as ELISA and RIA, are known in the art.

The term “antibody” herein is used in the broadest sense andspecifically includes full-length monoclonal antibodies, polyclonalantibodies, and, unless otherwise stated or contradicted by context,antigen-binding fragments, antibody variants, and multispecificmolecules thereof, so long as they exhibit the desired biologicalactivity. Generally, a full-length antibody is a glycoprotein comprisingat least two heavy (H) chains and two light (L) chains inter-connectedby disulfide bonds, or an antigen binding portion thereof. Each heavychain is comprised of a heavy chain variable region (abbreviated hereinas VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementarilydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen.

An “antigen-binding fragment” of an antibody is a molecule thatcomprises a portion of a full-length antibody which is capable ofdetectably binding to the antigen. Antigenbinding fragments includemultivalent molecules comprising one, two, three, or moreantigen-binding portions of an antibody, and single-chain constructswherein the VL and VH regions, or selected portions thereof, are joinedby synthetic linkers or by recombinant methods to form a functional,antigen-binding molecule.

The terms “antibody derivative” and “immunoconjugate” are usedinterchangeably herein to denote molecules comprising a full-lengthantibody or an antigen-binding fragment thereof, wherein one or moreamino acids are chemically modified, e.g., by alkylation, PEGylation,acylation, ester formation or amide formation or the like, e.g., forlinking the antibody to a second molecule. Exemplary modificationsinclude PEGylation, cysteinePEGylation, biotinylation, radiolabelling,and conjugation with a second agent, such as a detectable or cytotoxicagent.

A “multispecific molecule” comprises an antibody, or an antigen-bindingfragment thereof, which is associated with or linked to at least oneother functional molecule (e.g. another peptide or protein such asanother antibody or ligand for a receptor) to generate a molecule thatbinds to at least two different binding sites or target molecules.Exemplary multispecific molecules include bi-specific antibodies andantibodies linked to soluble receptor fragments or ligands.

A “humanized” antibody is a human/non-human chimeric antibody thatcontains a minimal sequence (CDR regions) derived from non-humanimmunoglobulin. Humanized antibodies are thus human immunoglobulins(recipient antibody) in which residues from a hypervariable region ofthe recipient are replaced by residues from a hypervariable region of anon-human species (donor antibody) such as mouse, rat, rabbit, ornon-human primate having the desired specificity, affinity, andcapacity. In some instances, FR residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are not found in the recipientantibody or in the donor antibody. These modifications are made tofurther refine antibody performance. In general, a humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableloops correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinsequence. The humanized antibody can optionally also comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity-determining region” or “CDR” (resi-dues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain;(Kabat et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and/or those residues from a “hypervariableloop” (residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy-chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987;196:901-917). Typically, the numbering of amino acid residues in thisregion is performed by the method described in Kabat et al., supra.Phrases such as “Kabat position”, “Kabat residue”, and “according toKabat” herein refer to this numbering system for heavy chain variabledomains or light chain variable domains. Using the Kabat numberingsystem, the actual linear amino acid sequence of a peptide may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include amino acid insertions (residue 52a,52b and 52c according to Kabat) after residue 52 of CDR H2 and insertedresidues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat)after heavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

“Framework region” or “FR” residues are those VH or VL residues otherthan the CDRs as herein defined.

“Corresponding” amino acid positions in two substantially identicalamino acid sequences are those aligned by any of the protein analysissoftware referred to herein, typically using default parameters.

An “isolated” molecule is a molecule that is the predominant species inthe composition wherein it is found with respect to the class ofmolecules to which it belongs (i.e., it makes up at least about 50% ofthe type of molecule in the composition and typically will make up atleast about 70%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or more of the species of molecule, e.g.,peptide, in the composition). Commonly, a composition of an antibodymolecule will exhibit 98%, 98%, or 99% homogeneity for antibodymolecules in the context of all present peptide species in thecomposition or at least with respect to substantially active peptidespecies in the context of proposed use.

The terms, “selectively neutralizes” and “selectively neutralizing”, asused herein, refer to an isolated and purified antibody (such as, butnot limited to a monoclonal antibody), or an antigen-binding fragmentthereof, that neutralizes selectively at least about 40%, at least about50%, or at least about 60% of a bioactivity of one or more IFN-α proteinsubtypes, but does not significantly neutralize at least one bioactivityof another IFN-α protein subtype, wherein the bioactivity can be, e.g.,activation of the MxA promoter and/or antiviral activity.

In the context of the present invention, “treatment” or “treating”refers to preventing, alleviating, managing, curing or reducing one ormore symptoms or clinically relevant manifestations of a disease ordisorder, unless contradicted by context. For example, “treatment” of apatient in whom no symptoms or clinically relevant manifestations of adisease or disorder have been identified is preventive or prophylactictherapy, whereas “treatment” of a patient in whom symptoms or clinicallyrelevant manifestations of a disease or disorder have been identifiedgenerally does not constitute preventive or prophylactic therapy.

The phrase “IFN-α-related condition or disease”, as used herein, refersto an abnormal condition, disease, or pre-clinical disease state thathas been linked with elevated levels of IFN-α in a patient's serum.Examples of such include, but are not limited to, lupus diseases ordisorders such as SLE, graft versus host disease (GVHD), type 1diabetes, AIDS (caused by human immunodeficiency virus (HIV)),autoimmune thyroiditis, and psoriasis. Methods for determining the levelof IFN-α are known in the art.

Humanized Anti-IFN-α Antibodies

The antibodies of the invention are humanized version of the anti-IFN-αmouse antibodies ACO-1 or ACO-2, variants thereof, and/orantigen-binding fragments thereof, characterized by particularfunctional and/or structural features or properties. Recombinantantibodies can be produced in suitable host cell lines by standardtechniques and be characterized by various assays to evaluate theirfunctional activities, as described below. In fact, it turns out thatIFN-alpha antibodies according to the present invention may be producedwith a significantly improved yield compared to IFN-alpha antibodiesthat were humanized in a traditional way.

According to the so-called “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against a library ofsuch known human variable-domain sequences or libraries of humangermline sequences. The human sequence that is closest to that of therodent can then be accepted as the human framework region for thehumanized antibody (Sims et al., J. Immunol. 1993; 151:2296 et seq.;Chothia et al, Chothia and Lesk, J. Mol. Biol. 1987; 196:901-917).Another method uses a particular framework region derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies.

Preferred framework sequences for use in the antibodies of the inventionare those that are structurally similar to the framework sequences usedby ACO-1 or ACO-2. Thus, in one embodiment, the invention provides ahumanized ACO-1 or ACO-2 anitbody comprising VH framework residuesderived from a human VH1_(—)46 gene and a human JH4 gene, and VLframework residues derived from a human VKIII_L6 gene and a human JK2gene, and specifically binds human IFN-α.

Example 1 below describes the design of an exemplary humanized ACO-1antibody, hzACO-1, comprising such framework sequences.

Functional Properties

The humanized antibodies of the invention bind specifically to IFN-αsubtypes A, 2, B2, C, F, G, H2, I, J1, K, 4a, 4b, and WA (Table 2). Inone embodiment, a humanized ACO-1 or ACO-2 antibody of the inventionbinds to an IFN-α protein subtype such as IFN-αA with high affinity, forexample with a KD of about 10⁻⁷ M or less, a KD of about 10⁻⁸ M or less,a KD of about 5×10⁻⁹ M or less, or a KD of about 2×10⁻⁹ M or less. Inone embodiment, the humanized antibody is a hzACO-1 variant which bindsto IFN-αA, IFN-αF, and/or another IFN-α protein subtypes with anaffinity comparable to or higher than that of hzACO-1.

TABLE 2 Kinetic parameters of hzACO-1 for a range of human IFN-αsubtypes Sample ka (1/Ms) kd (1/s) KD (M) hIFN-αA 2.97E+05 3.94E−041.33E−09 hIFN-α1 No binding — — hIFN-α2 3.58E+05 3.51E−04 9.81E−10hIFN-α4b 3.74E+05 6.22E−04 1.67E−09 hIFN-αG 4.63E+05 4.26E−04 9.20E−10hIFN-αH2 3.78E+05 1.21E−03 3.21E−09 hIFN-αI 7.23E+05 2.03E−03 2.81E−09hIFN-αJ1 6.81E+05 3.27E−03 4.81E−09 hIFN-αWA 7.09E+05 2.91E−03 4.10E−09hIFN-α4a 3.33E+04 1.15E−04 3.45E−09 hIFN-αC 7.19E+05 7.53E−04 1.05E−09hIFN-αK 5.74E+05 8.27E−04 1.44E−09

For example, the ratio between the KD of the hzACO-1 variant and the KDof hzACO-1 to a IFN-αA protein subtype can be about 1.0, about 0.8,about 0.7, or about 0.6. In another embodiment, the humanized antibodyis a hzACO-1 variant which binds to IFN-αA, IFN-αF, and/or another IFN-αprotein subtype with an affinity comparable to or higher than that ofrecombinantly produced ACO-1. In another embodiment, the humanizedantibody is a hzACO-1 variant which binds to IFN-αA, IFN-αF, and/oranother IFN-α protein subtypes with an affinity comparable to or higherthan that of hybridoma-produced ACO-1.

Furthermore, the humanized antibodies of the invention are capable ofselectively neutralizing a bioactivity of one or more IFN-α proteinsubtypes. For example, a humanized ACO-1 or ACO-2 variant can be capableof selectively neutralizing at least about 40%, at least about 50%, orat least about 60% of a bioactivity of IFN-α protein subtypes A, 2, B2,C, F, G, H2, I, J1, K, 4a, 4b, or WA, or any combination thereof, ascompared to a control. In a particular embodiment, the humanizedantibody does not significantly neutralize the bioactivity of IFN-αsubtypes D and/or 1. Exemplary bioactivities include, but are notlimited to, activation of the MxA promoter, antiviral activity, or both.The ability of a humanized antibody to neutralize such IFN-α bioactivitycan be evaluated using, e.g., the reporter gene (RG) an cytopathicinhibition (CPE) assays described herein. In one embodiment, thehumanized antibody is a hzACO-1 variant which has an IC50 comparable toor lower than the IC50 of hzACO-1 in an RG assay. In a specificembodiment, the hzACO-1 variant has a lower IC50 than hzACO-1 in an RGassay.

In one embodiment, the humanized antibodies of the invention competewith and/or bind to the same epitope on an IFN-α protein subtype asACO-1 and/or ACO-2. Such antibodies can be identified based on theirability to cross-compete with ACO-1 and/or ACO-2 in standard IFN-αbinding assays as described herein. The ability of a test humanizedantibody antibody to inhibit the binding of ACO-1 or ACO-2 to one ormore IFN-α protein subtypes demonstrates that the test antibody cancompete with ACO-1 or ACO-2 for binding to IFN-α and thus can bind tothe same epitope on the IFN-α protein subtype as ACO-1 and/or ACO-2(WO02066649 and WO2005059106). In a particular embodiment, the humanizedantibody binds to a different human IFN-α epitope than any of the murinemonoclonal antibodies 9F3, and MMHA-1, -2, -3, -6, -8, -9, -11, -13, and-17 (PBL Biomedical Laboratories, NJ, USA) and/or human monoclonalantibodies 13H5, 13H7, and 7H9, and/or cross-competes more with ACO-1 orACO-2 than with one or more of the listed murine and human monoclonalantibodies.

In one embodiment, hzACO-1, hzACO-2, hzACO-1 variants or hzACO-2variants provided by the invention have an immunogenicity comparable toor lower than that of a humanized ACO-1 or ACO-2 antibody comprisingmurine CDRs according to Kabat (Kabat ACO-1). Immunogenicity of ahumanized antibody can be evaluated by, e.g., one or more of the methodsdescribed in Wadwha et al., Dev Biol (Basel). 2005; 122:155-70), whichis hereby incorporated by reference in its entirety.

In further aspect, the humanized antibodies of the invention are stablein a formulation suitable for administration to a human patient. In oneembodiment, the humanized ACO-1 or ACO-2 antibody according to theinvention is at least as stable as the IFN-alpha antibodies humanized ina traditional way comprising the full length murine Kabat sequences.

Stability of an antibody can be evaluated using known methods in theart, including the thermofluor analyses described in Example 11.

Preferred humanized antibodies of the invention exhibit at least one,more preferably two, three, four, five or more, of the followingproperties: (a) bind specifically to IFN-α subtypes A, 2, B2, C, F, G,H2, I, J1, K, 4a, 4b, and WA; (b) selectively neutralize one or morebioactivities of IFN-α protein subtypes A, 2, B2, C, F, G, H2, I, J1, K,4a, 4b, or WA; any combination thereof, or all thereof; (c) do notsignificantly neutralize a bioactivity of IFN-α1 or D; (d) compete withand/or bind to the same epitope on an IFN-α protein subtype as ACO-1and/or ACO-2; (e) compete more with ACO-1 or ACO-2 than with any of 9F3,13H5, 13H7, and 7H9; (f) are less likely to elicit an immune responsethan a hzACO-1 or hzACO-2 antibody comprising murine CDRs according toKabat; (g) are stable in pharmaceutical formulations; and (h) binds toat least one of IFN-α protein subtypes A, 2, B2, C, F, G, H2, I, J1, K,4a, 4b, or WA with a KD of 10⁻⁸ M or less.

Structural Properties

Preferred antibodies of the invention are humanized versions of themurine monoclonal antibodies ACO-1 and ACO-2. Such antibodies can beproduced, isolated, and structurally and functionally characterized asdescribed in the Examples. Full-length, variable region, and Kabat CDRsequences of ACO-1, hzACO-1 and ACO-2 are set forth in Table 3 anddescribed in FIGS. 1-3.

TABLE 3  Sequence numbering for primers, protein and antibodies SequenceSEQ Antibody portion composition Sequence ID NO: ACO-1 VH Protein  1VH1_46/JH4 Protein  2 hzACO-1 VH Protein  3 ACO-1 VL Protein  4VKIII_L6/JK2 Protein  5 hzACO-1 VL Protein  6 ACO-2 VH Protein  7J558.33/D/JH3_1 Protein  8 ACO-2 VL Protein  9 ae4/JK4_1 Protein 10PCR Primer ACO-1 cloning DNA 11 PCR Primer ACO-1 cloning DNA 12 ACO-1 VHDNA 13 ACO-1 VL DNA 14 ACO-1 CDR_H1 Protein NYWMH 15 ACO-1 CDR_H2Protein EINPSHGRTIYNENFKS 16 ACO-1 CDR_H3 Protein GGLGPAWFAY 17ACO-1 CDR_L1 Protein SAGSSVDSSYLY 18 ACO-1 CDR_L2 Protein STSNLAS 19ACO-1 CDR_L3 Protein HQWSSYPFT 20 hzACO-1 CDR_H2 ProteinEINPSHGRTIYAQKFQG 21 ACO-2 CDR_H1 Protein SYWMH 22 ACO-2 CDR_H2 ProteinEINPSHGRTSYNENFKS 23 ACO-2 CDR_L1 Protein SAGSSVGSSYFY 24 ACO-2 CDR_L2Protein GTSNLAS 25 PCR Primer ACO-2 cloning DNA 26PCR Primer ACO-2 cloning DNA 27 ACO-2 VH DNA 28 ACO-2 VL DNA 29 hIFN-α8Protein 30 hzACO-1 Fab HC Protein 31 hzACO-1 LC Protein 32

The sequences of hzACO-1 CDR H1, H3, L1, L2, and L3 are identical to thecorresponding ACO-1 sequences. ACO-2 CDR H3 and L3 are identical to thecorresponding ACO-1 CDR sequences. The amino acids shown in italics inthe hzACO-1 CDR_H2 sequence correspond to the human frameworksequence—in traditionally humanized antibodies the full length Kabatsequence correspond to the ACO-1 CDR_H2 sequence, where all amino acidsare derived from the murine antibody.

In one aspect, the invention provides humanized versions of murine ACO-1and ACO-2 antibodies with fewer donor residues than the Kabat CDRs,i.e., fewer murine residues than a humanized ACO-1 or ACO-2 antibodyproduced by grafting of the kabat CDRs.

In one embodiment, the humanized antibody specifically binds human IFN-αand is a humanized version of murine antibody ACO-1 or ACO-2, or of acombination thereof, comprising fewer donor amino acid residues than themurine complementary determining regions (CDRs) according to Kabat. TheCDR H2 sequence may, for example, comprise fewer donor amino acidresidues than those corresponding to Kabat residues 50-65, 50-64, 50-63,50-62, 50-61, or 50-60. The CDR H2 donor residues may comprise Kabatresidues 50-59. Additionally or alternatively, the CDR H2 donor aminoacid residues may consist of Kabat residues 50-59. Kabat residues 50-59correspond to residues 1-11 of SEQ ID NOS:16, 21, and 23. In oneembodiment, the remaining VH CDRs may comprise or consist of the KabatCDRs (see FIGS. 1-3), i.e., a CDR H1 sequence comprising donor aminoacid residues corresponding to Kabat residues 31-35, and a CDR H3sequence comprising donor amino acid residues corresponding to Kabatresidues 95-102.

In one embodiment, the humanized ACO-1 or ACO-2 antibody may comprise aCDR L1 comprising donor amino acid residues corresponding to Kabatresidues 24-34 of the variable region of the ACO-1 light chain (VL), aCDR L2 comprising donor amino acid residues corresponding to Kabatresidues 50-56 of the ACO-1 VL region, and a CDR L3 comprising donoramino acid residues corresponding to Kabat residues 89-97 of the ACO-1VL region (SEQ ID NO:4) or ACO-2 VL region (SEQ ID NO:9). Additionallyor alternatively, the antibody may comprise CDR L1 donor amino acidresidues consisting of Kabat residues 24-34, CDR L2 donor residuesconsisting of Kabat residues 50-56, and CDR L3 donor amino acid residuesconsisting of Kabat residues 89-97. The corresponding amino acidsequences are shown in Table 3.

In one aspect, the invention provides specific humanized ACO-1antibodies. The humanized ACO-1 antibody specifically binds human IFN-α,and comprises VH CDR sequences substantially identical to the sequencesof Kabat residues 31-35, 50-65, and 95-102 of SEQ ID NO:3, with anoptional N31S mutation. The antibody may, e.g., comprise a CDR H1sequence comprising SEQ ID NO:15; a CDR H2 sequence comprising SEQ IDNO:21; and a CDR H3 sequence comprising SEQ ID NO:17. Additionally oralternatively, the antibody may comprise a CDR H1 sequence consisting ofSEQ ID NO:15; a CDR H2 sequence consisting of SEQ ID NO:21; and a CDR H3sequence consisting of SEQ ID NO:17. In one embodiment, the humanizedACO-1 comprises VH framework residues derived from a human VH1_(—)46gene and/or a human JH4 gene, preferably both. In a specific embodiment,the humanized antibody comprises a VH sequence corresponding to SEQ IDNO:3.

The humanized ACO-1 antibody may further comprise VL CDR sequencessubstantially identical to the sequences of Kabat residues 24-34, 50-56,and 89-97 of SEQ ID NO:6. The antibody may, e.g., comprise a CDR_L1sequence comprising SEQ ID NO:18; a CDR_L2 sequence comprising SEQ IDNO:19; and a CDR_L3 sequence comprising SEQ ID NO:20. Additionally oralternatively, the may comprise a CDR_L1 sequence consisting of SEQ IDNO:18; a CDR_L2 sequence consisting of SEQ ID NO:19; and a CDR_L3sequence consisting of SEQ ID NO:20. In one embodiment, the humanizedACO-1 antibody comprises VL framework residues derived from a humanVKIII_L6 gene and/or a human JK2 gene, preferably both. In a specificembodiment, the humanized antibody comprises a VL sequence correspondingto SEQ ID NO:6.

In one aspect, the invention provides an antibody comprising the CDRsequences of ACO-2. The antibody can specifically bind human IFN-α andcomprises VH CDR sequences substantially identical to the sequences ofKabat residues 31-35, 50-59, and 95-102 of SEQ ID NO:7. In oneembodiment, the antibody comprises a CDR_H1 sequence comprising SEQ IDNO:22; a CDR_H2 sequence comprising SEQ ID NO:23; and a CDR_H3 sequencecomprising SEQ ID NO:17. In an additional or alternative embodiment, theantibody comprises a CDR_H1 sequence consisting of SEQ ID NO:22; aCDR_H2 sequence consisting of SEQ ID NO:23; and a CDR_H3 sequenceconsisting of SEQ ID NO:17. The antibody may further comprise VL CDRsequences substantially identical to the sequences of residues Kabatresidues 24-34, 50-56, and 89-97 of SEQ ID NO:9. In one embodiment, theantibody comprises a CDR_L1 sequence comprising SEQ ID NO:24; a CDR_L2sequence comprising SEQ ID NO:25; and a CDR_L3 sequence comprising SEQID NO:20. Additionally or alternatively, the antibody comprises a CDR_L1sequence consisting of SEQ ID NO:24; a CDR_L2 sequence consisting of SEQID NO:25; and a CDR_L3 sequence consisting of SEQ ID NO:20. The antibodymay, in one aspect, be a humanized ACO-2 antibody.

A humanized ACO-1 or ACO-2 antibody may further comprise at least aportion of a human Fc-region (unless the antibody is an antigen-bindingfragment not comprising any Fc-portion). Typically, the size of theFc-region is selected to achieve the desired pharmacokinetic propertiesof the antibody; the larger Fc-portion, the slower clearance. In oneembodiment, the humanized antibody is a full-length antibody, preferablycomprising an IgG4 isotype Fc-region. In a particular embodiment, theIgG4 Fc-region comprises an S241P mutation, with numbering according toKabat; corresponding to residue 228 per the EU numbering system (EdelmanG. M. et AL., Proc. Natl. Acad. USA 63, 78-85 (1969)).

Given that both ACO-1 and ACO-2 can bind to IFN-α and are similar, thehumanized VH and VL sequences can be “mixed and matched” to create otheranti-IFN-α binding molecules of the invention. IFN-α binding of such“mixed and matched” antibodies can be tested using the binding assaysdescribed herein (e.g. flow cytometry, Biacore® analysis, ELISAs) and/orusing one or more functional assays as described herein. Preferably,when VH and VL chains are mixed and matched, a VH sequence from aparticular VH/VL pairing is replaced with a structurally similar VHsequence. Likewise, a VL sequence from a particular VH/VL pairing ispreferably replaced with a structurally similar VL sequence.

Accordingly, in one aspect, the invention provides an humanizedmonoclonal antibody, or antigen binding portion thereof, comprising: (a)a VH region comprising ACO-1 or ACO-2 VH CDRs and (b) a VL regioncomprising ACO-1 or ACO-2 VL CDRs; wherein the antibody specificallybinds IFN-α. Preferred heavy and light chain combinations include: (a) aVH region comprising SEQ ID NOS:15-17, optionally omitting some or allof the 5 C-terminal amino acids of SEQ ID NO:16, and (b) a light chainvariable region comprising SEQ ID NOS:18-20; (a) a VH region comprisingSEQ ID NOS:15-17, optionally omitting some or all of the 5 C-terminalamino acids of SEQ ID NO:16, and (b) a light chain variable regioncomprising SEQ ID NOS:24, 25, and 20; (a) a VH region comprising SEQ IDNOS:22, 23, and 17, optionally omitting some or all of the 5 C-terminalamino acids of SEQ ID NO:23, and (b) a light chain variable regioncomprising SEQ ID NOS:18-20; and (a) a VH region comprising SEQ IDNOS:22, 23, and 17, optionally omitting some or all of the 5 C-terminalamino acids of SEQ ID NO:23, and (b) a light chain variable regioncomprising SEQ ID NOS:24, 25, and 20. Other preferred heavy and lightchain combinations include (a) a VH region comprising the sequence ofSEQ ID NO:3 and (b) a VL region comprising the amino acid sequence ofSEQ ID NO:4; (a) a VH comprising SEQ ID NOS:15, 21, and 17, and (b) a VLcomprising SEQ ID NOS: 18-20; and (a) a VH comprising SEQ ID NOS:15, 21,and 17, and (b) a VL comprising SEQ ID NOS:24, 25, and 20.

In another aspect, the invention provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and/or CDR3s of ACO-1 or ACO-2,or combinations thereof. Given that each of these antibodies can bind toIFN-α and that antigen-binding specificity is provided primarily by theCDR1, 2 and 3 regions, the CDR H1, H2 and H3 sequences and CDR L1, L2and L3 sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and matched, although each antibody can containa CDR H1, H2 and H3 and a CDR L1, L2 and L3) to create other anti-IFN-αbinding molecules of the invention. IFN-α-binding of such “mixed andmatched” antibodies can be tested using the binding assays describedbelow and in the Examples (e.g. flow cytometry, Biacore® analysis, orELISAs). Preferably, when VH CDR sequences are mixed and matched, theCDR H1, H2 and/or H3 sequence from a particular VH sequence is replacedwith a structurally similar CDR sequence(s). Likewise, when VL CDRsequences are mixed and matched, the CDR L1, L2 and/or L3 sequence froma particular VL sequence preferably is replaced with a structurallysimilar CDR sequence(s). For example, the CDRs of ACO-1 and ACO-2 sharesubstantial structural similarity and therefore are amenable to mixingand matching.

Accordingly, in another aspect, the invention provides a humanizedmonoclonal antibody, or antigen binding portion thereof comprising: (a)a CDR H1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS:15 and 22; (b) a CDR H2 comprising an aminoacid sequence selected from the group consisting of at least residues1-12 of SEQ ID NOS:16 and 23, (c) a CDR H3 comprising SEQ ID NO:17; (d)a CDR L1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS:18 and 24; (e) a CDR L2 comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS:19 and25; and (f) a CDR L3 comprising SEQ ID NO:20; wherein the antibodyspecifically binds IFN-α.

In a preferred embodiment, the antibody comprises: (a) a CDR H1comprising SEQ ID NO:15; (b) a CDR H2 comprising at least residues 1-12of SEQ ID NO:16; (c) a CDR H3 comprising SEQ ID NO:17; (d) a CDR L1comprising SEQ ID NO:18; (e) a CDR L2 comprising SEQ ID NO:19; and (f) aCDR L3 comprising SEQ ID NO:20.

In another preferred embodiment, the antibody comprises: (a) a CDR H1comprising SEQ ID NO: 22; (b) a CDR H2 comprising at least residues 1-12of SEQ ID NO:23; (c) a CDR H3 comprising SEQ ID NO:17; (d) a CDR L1comprising SEQ ID NO:24; (e) a CDR L2 comprising SEQ ID NO:25; and (f) aCDR L3 comprising SEQ ID NO:20.

In a preferred embodiment, the antibody comprises: (a) a CDR H1comprising SEQ ID NO:15; (b) a CDR H2 comprising SEQ ID NO:21; (c) a CDRH3 comprising SEQ ID NO:17; (d) a CDR L1 comprising SEQ ID NO:18; (e) aCDR L2 comprising SEQ ID NO:19; and (f) a CDR L3 comprising SEQ IDNO:20.

In a preferred embodiment, the antibody comprises: (a) a CDR H1comprising SEQ ID NO:22; (b) a CDR H2 comprising at least residues 1-12of SEQ ID NO:16; (c) a CDR H3 comprising SEQ ID NO:17; (d) a CDR L1comprising SEQ ID NO:18; (e) a CDR L2 comprising SEQ ID NO:19; and (f) aCDR L3 comprising SEQ ID NO:20.

In a preferred embodiment, the antibody comprises: (a) a CDR H1comprising SEQ ID NO:15; (b) a CDR H2 comprising at least residues 1-12of SEQ ID NO:16; (c) a CDR H3 comprising SEQ ID NO:17; (d) a CDR L1comprising SEQ ID NO:24; (e) a CDR L2 comprising SEQ ID NO:19; and (f) aCDR L3 comprising SEQ ID NO:20.

In a preferred embodiment, the antibody comprises: (a) a CDR H1comprising SEQ ID NO:15; (b) a CDR H2 comprising at least residues 1-12of SEQ ID NO:16; (c) a CDR H3 comprising SEQ ID NO:17; (d) a CDR L1comprising SEQ ID NO:18; (e) a CDR L2 comprising SEQ ID NO:25; and (f) aCDR L3 comprising SEQ ID NO:20.

Humanized Anti-IFN-α Antibody Variants

Though an antibody variant or derivative typically has at least onealtered property as compared to the parent antibody, antibody variantsor derivatives can retain one, some, most, or all of the functionalproperties of the patent anti-IFN-α antibody, including, but not limitedto: (a) bind specifically to IFN-α subtypes A, 2, B2, C, F, G, H2, I,J1, K, 4a, 4b, and WA; (b) selectively neutralize one or morebioactivities of IFN-α protein subtypes A, 2, B2, C, F, G, H2, I, J1, K,4a, 4b, or WA; any combination thereof, or all thereof; (c) do notsignificantly neutralize a bioactivity of IFN-α1 or D; (d) compete withand/or bind to the same epitope on an IFN-α protein subtype as ACO-1and/or ACO-2; (e) compete more with ACO-1 or ACO-2 than with any of 9F3,13H5, 13H7, and 7H9; (f) are less likely to elicit an immune responsethan a hzACO-1 or hzACO-2 antibody comprising murine CDRs according toKabat; (g) are stable in pharmaceutical formulations; and (h) binds toat least one of IFN-α protein subtypes A, 2, B2, C, F, G, H2, I, J1, K,4a, 4b, or WA with a KD of 10⁻⁸ M or less. Any combination of theabove-described functional features, and/or the functional features asdescribed in the Examples, may be exhibited by an antibody of theinvention.

In certain embodiments, a humanized antibody of the invention comprisesa VH region comprising CDR H1-H3 sequences and a VL region comprisingCDR L1-L3 sequences, wherein one or more of these CDR sequences comprisespecified amino acid sequences based on the preferred antibodiesdescribed herein; ACO-1 and ACO-2, or conservative modificationsthereof, and wherein the antibodies have retained or improved thedesired functional properties of the anti-IFN-α antibodies of theinvention. Accordingly, the invention provides an isolated monoclonalantibody, or antigen-binding fragment thereof, comprising a heavy chainvariable region comprising CDR H1, CDR H2, and CDR H3 sequences and alight chain variable region comprising CDR H1, CDR H2, and CDR H3sequences, wherein: (a) a CDR H1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOS:15 and 22, andconservative modifications thereof; (b) a CDR H2 comprising an aminoacid sequence selected from the group consisting of at least residues1-12 of SEQ ID NOS:16 and 23, and conservative modifications thereof,(c) a CDR H3 comprising SEQ ID NO:17, and conservative modificationsthereof; (d) a CDR L1 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOS:18 and 24, and conservativemodifications thereof; (e) a CDR L2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOS:19 and 25, andconservative modifications thereof; and (f) a CDR L3 comprising SEQ IDNO:20, and conservative modifications thereof; wherein the antibodyspecifically binds IFN-α.

Thus, one or more amino acid residues within the CDR or FR regions of anantibody of the invention can be replaced with other amino acid residuesfrom the same side chain family and the altered antibody can be testedfor retained function (i.e., the functions set forth in (c), (d) and (e)above) using the functional assays described herein.

The functional properties of the antibody variants can be assessed usingstandard assays available in the art and/or described herein. Forexample, the ability of the antibody to bind IFN-α can be determinedusing standard binding and biological effects (e.g. reporter gene)assays, such as those set forth in the Examples (e.g., Biacore®analysis, or ELISAs).

Variable Region Modifications

In one aspect, the invention provides a humanized ACO-1 or ACO-2antibody with mutations in the CDR or framework regions.

Specific exemplary mutations in humanized ACO-1 and their identificationare described in Examples 2 and 3 (see, also FIGS. 1 and 3). Theseinclude both back-mutations; introducing ACO-1 residues into humanizedACO-1, as well as mutations where ACO-2-derived residues are introducedinto humanized ACO-1. Exemplary back-mutations in the hzACO-1 VH (SEQ IDNO:3) include V5Q, M69L, R71V, T73K, S761, and V78A, as well as anycombination thereof, using Kabat numbering. Exemplary back-mutations inthe hzACO-1 VL (SEQ ID NO:6) include E1Q, L47W, I58V, and F71Y, as wellas any combination thereof. Exemplary ACO-2-derived mutations in thehzACO-1 VH (SEQ ID NO:3) include T28S, N31S, I58S, S76N, T771, and A93V,as well as any combination thereof. Exemplary ACO-2 derived mutations inthe hzACO-1 VL (SEQ ID NO:6) include D29G, L33F, and 550G, as well asany combination thereof.

Further, various hzACO-1 VH and VL variant sequences can be “mixed andmatched” with variant sequences or parent sequences to create a libraryof hzACO-1 variants of the invention. IFN-α-binding of such “mixed andmatched” antibodies can be tested using the binding assays describedherein (e.g., Biacore® analysis, ELISAs) and/or using one or morefunctional assays as described herein.

In one embodiment, the invention provides a humanized antibody thatspecifically binds human IFN-α and contains a variable domain having,incorporated into a human antibody variable domain, amino acids from adonor non-human antibody that binds human IFN-α, comprising a donorantibody amino acid residue at one or more sites selected from 5, 28,31, 58, 69, 71, 73, 76, 78, 79, and 93 in the heavy chain variabledomain.?

In one embodiment, the invention provides a humanized antibody thatspecifically binds human IFN-α and contains a variable domain having,incorporated into a human antibody variable domain, amino acids from adonor non-human antibody that binds human IFN-α, comprising a donorantibody amino acid residue at one or more sites selected from 1, 29,33, 47, 50, 58, and 71 in the light chain variable domain.

In one embodiment, the invention provides a humanized ACO-1 antibodythat specifically binds human IFN-α and contains a variable domainhaving, incorporated into a human antibody variable domain, CDRsequences from ACO-1 that bind human IFN-α, and further comprising ACO-2amino acid residues at one or more sites selected from 28, 31, 58, 76,77, 78, 79, and 93 in the heavy chain variable domain. In a specificembodiment, the ACO-2 amino acid residues are at one or more sitesselected from 28, 31, and 93.

In one embodiment, the invention provides a humanized ACO-1 antibodythat specifically binds human IFN-α and contains a variable domainhaving, incorporated into a human antibody variable domain, CDRsequences from ACO-1 that bind human IFN-α, and further comprising ACO-2amino acid residues at one or more sites selected from 29, 33, and 50 inthe light chain variable domain.

In one embodiment, the invention provides a hzACO-1 variant thatspecifically binds human IFN-α, and comprises VH CDR sequencessubstantially identical to the sequences of Kabat residues 31-35, 50-65,and 95-102 of SEQ ID NO:3, with an N31S mutation. The antibody may,e.g., comprise a CDR H1 sequence comprising SEQ ID NO:15 with an N31Smutation; a CDR H2 sequence comprising SEQ ID NO:21; and a CDR H3sequence comprising SEQ ID NO:17. Additionally or alternatively, theantibody may comprise a CDR H1 sequence consisting of SEQ ID NO:15 withan N31S mutation; a CDR H2 sequence consisting of SEQ ID NO:21; and aCDR H3 sequence consisting of SEQ ID NO:17. In one embodiment, thehumanized ACO-1 comprises VH framework residues derived from a humanVH1_(—)46 gene and/or a human JH4 gene, preferably both. In a specificembodiment, the humanized antibody comprises a VH sequence correspondingto SEQ ID NO:3, with an N to S mutation at Kabat position 31. As shownin the Examples, an N31S mutation in hzACO-1 increased the bindingaffinity to IFN-αA to levels comparable to that of ACO-1, and increasedthe stability at pH3.5 and 4.5. Moreover, since residue 31 is in a“donor” CDR residue, the mutation does not introduce a further murineresidue into the hzACO-1 sequence, thus not increasing the risk for animmune response against the antibody when administered to a human. OtherCDR mutations with the same advantage include D29G and S50G in thehzACO-1 VL.

In one embodiment, the invention provides a hzACO-1 variant thatspecifically binds human IFN-α, and comprises VH CDR sequencessubstantially identical to the sequences of Kabat residues 31-35, 50-65,and 95-102 of SEQ ID NO:3, with a T28S mutation. The antibody may, e.g.,comprise a CDR H1 sequence comprising SEQ ID NO:15; a CDR H2 sequencecomprising SEQ ID NO:21; and a CDR H3 sequence comprising SEQ ID NO:17.Additionally or alternatively, the antibody may comprise a CDR H1sequence consisting of SEQ ID NO:15; a CDR H2 sequence consisting of SEQID NO:21; and a CDR H3 sequence consisting of SEQ ID NO:17. In oneembodiment, the humanized ACO-1 comprises VH framework residues derivedfrom a human VH1_(—)46 gene, further comprising a T28S mutation. In aspecific embodiment, the humanized antibody comprises a VH sequencecorresponding to SEQ ID NO:3, with an T to S mutation at Kabat position28. As shown in the Examples, a T28S mutation in hzACO-1 increased thebinding affinity to IFN-αA to levels comparable to that of ACO-1, andincreased stability at pH 3.5 and 4.5.

In one embodiment, the invention provides a hzACO-1 variant thatspecifically binds human IFN-α, and comprises VH CDR sequencessubstantially identical to the sequences of Kabat residues 31-35, 50-65,and 95-102 of SEQ ID NO:3, with an A93V mutation. The antibody may,e.g., comprise a CDR H1 sequence comprising SEQ ID NO:15; a CDR H2sequence comprising SEQ ID NO:21; and a CDR H3 sequence comprising SEQID NO:17. Additionally or alternatively, the antibody may comprise a CDRH1 sequence consisting of SEQ ID NO:15; a CDR H2 sequence consisting ofSEQ ID NO:21; and a CDR H3 sequence consisting of SEQ ID NO:17. In oneembodiment, the humanized ACO-1 comprises VH framework residues derivedfrom a human VH1_(—)46 gene and a human JH4 gene, further comprising anA93V mutation. In a specific embodiment, the humanized antibodycomprises a VH sequence corresponding to SEQ ID NO:3, with an A to Vmutation at Kabat position 93. As shown in the Examples, an A93Vmutation in hzACO-1 increased the binding affinity to IFN-αA to levelscomparable to that of ACO-1, increased the potency for inhibition ofIFN— effects as measured in the RG assay, and increased stability atpH3.5 and 4.5.

In one embodiment, the invention provides a hzACO-1 variant thatspecifically binds human IFN-α, and comprises VH CDR sequencessubstantially identical to the sequences of Kabat residues 31-35, 50-65,and 95-102 of SEQ ID NO:3, further comprising a mutation in one of theVH CDR sequences, wherein the mutation is not in Kabat residue 58. Theantibody may, e.g., comprise VH CDRs consisting of Kabat residues 31-35,50-65, and 95-102 of SEQ ID NO:3, further comprising a mutation in oneof the VH CDR sequences, wherein Kabat residue 58 is I. In oneembodiment, the humanized ACO-1 comprises VH framework residues derivedfrom a human VH1_(—)46 gene and a human JH4 gene. As shown in theExamples, an I58S mutation in hzACO-1 substantially decreased thebinding affinity to IFN-αA and decreased the potency for inhibition ofIFN-effects as measured in the RG assay.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

Fc Modifications

In addition or as an alternative to modifications made within theframework or CDR regions, antibodies of the invention may be engineeredto include modifications within the Fc region, typically to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, protein stability and/orantigen-dependent cellular cytotoxicity, or lack thereof. Furthermore,an antibody of the invention may be chemically modified (e.g., one ormore chemical moieties can be attached to the antibody) or be modifiedto alter its glycosylation, again to alter one or more functionalproperties of the antibody. Each of these embodiments is described infurther detail below. The residues in the Fc region are numberedaccording to Kabat.

If desired, the class of an antibody may be “switched” by knowntechniques. Such techniques include, e.g., the use of direct recombinanttechniques (see e.g., U.S. Pat. No. 4,816,397) and cell-cell fusiontechniques (see e.g., U.S. Pat. No. 5,916,771). For example, an antibodythat was originally produced as an IgM molecule may be class switched toan IgG antibody. Class switching techniques also may be used to convertone IgG subclass to another, e.g., from IgG1 to IgG2. Thus, the effectorfunction of the antibodies of the invention may be changed by isotypeswitching to, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody for various therapeutic uses. Exemplary cDNA sequences forconstant regions are available via, e.g., GenBank, each of whichincorporated by reference in its entirety, are as follows:

-   -   Human IgG1 constant heavy chain region: GenBank accession No.:        J00228;    -   Human IgG2 constant heavy chain region: GenBank accession No.:        J00230;    -   Human IgG3 constant heavy chain region: GenBank accession No.:        X04646;    -   Human IgG4 constant heavy chain region: GenBank accession No.:        K01316; and    -   Human kappa light chain constant region: GenBank accession No.:        J00241.

In one embodiment, the hinge region of CH₁ is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al. In another embodiment,the antibody is modified to increase its biological half life. Variousapproaches are possible. For example, one or more of the followingmutations can be introduced: T252L, T254S, T256F, as described in U.S.Pat. No. 6,277,375 to Ward. Alternatively, to increase the biologicalhalf life, the antibody can be altered within the CH1 or CL region tocontain a salvage receptor binding epitope taken from two loops of a CH2domain of an Fc region of an IgG, as described in U.S. Pat. Nos.5,869,046 and 6,121,022 by Presta et al. In yet other embodiments, theFc region is altered by replacing at least one amino acid residue with adifferent amino acid residue to alter the effecter function(s) of theantibody. For example, one or more amino acids selected from amino acidresidues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced witha different amino acid residue such that the antibody has an alteredaffinity for an effector ligand but retains the antigen-binding abilityof the parent antibody. The effector ligand to which affinity is alteredcan be, for example, an Fc receptor or the C1 component of complement.This approach is described in further detail in U.S. Pat. Nos. 5,624,821and 5,648,260, both to Winter et al. In another example, one or moreamino acids selected from amino acid residues 329, 331 and 322 can bereplaced with a different amino acid residue such that the antibody hasaltered C1q binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 by Idusogie et al. In another example, one or moreamino acid residues within amino acid positions 231 and 239 are alteredto thereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al. In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcy receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcyRI, FcyRII, FcyRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcRIII. Additionally, thefollowing combination mutants were shown to improve FcyRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

The constant region may further be modified to stabilize the antibody,e.g., to reduce the risk of a bivalent antibody separating into twomonovalent VH-VL fragments. For example, in an IgG4 constant region,residue S241 may be mutated to a proline (P) residue to allow completedisulphide bridge formation at the hinge (see, e.g., Angal et al., Mol.Immunol. 1993; 30:105-8).

Glycosylation Modifications

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence.

Antigen-binding Fragments

The anti-IFN-α antibodies of the invention may be prepared asfull-length antibodies or antigen-binding fragments thereof. Examples ofantigen-binding fragments include Fab, Fab′, F(ab)₂, F(ab′)2, F(ab)₃, Fv(typically the VL and VH domains of a single arm of an antibody),single-chain Fv (scFv; see e.g., Bird et al., Science 1988; 242:423-426;and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd (typically the VHand CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VhH,and V-NAR domains; monovalent molecules comprising a single VH and asingle VL chain; minibodies, diabodies, triabodies, tetrabodies, andkappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10:949-57); camelIgG; IgNAR; as well as one or more isolated CDRs or a functionalparatope, where the isolated CDRs or antigen-binding residues orpolypeptides can be associated or linked together so as to form afunctional antibody fragment. Various types of antibody fragments havebeen described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol2005; 23:1126-1136; WO2005040219, and published U.S. Patent Applications20050238646 and 20020161201.

Antibody fragments can be obtained using conventional recombinant orprotein engineering techniques, and the fragments can be screened forantigen-binding or other function in the same manner as are intactantibodies.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of full-length antibodies (see, e.g., Morimoto et al., Journalof Biochemical and Biophysical Methods, 24:107-117 (1992); and Brennanet al., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Alternatively, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)2 fragments (Carter et al., Bio/Technology, 10:163-167(1992)). According to another approach, F(ab′)2 fragments can beisolated directly from recombinant host cell culture. In otherembodiments, the antibody of choice is a single-chain Fv fragment(scFv). See WO 1993/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.5,587,458. The antibody fragment may also be a “linear antibody”, e.g.,as described in U.S. Pat. No. 5,641,870, for example. Such linearantibody fragments may be monospecific or bispecific.

Multispecific Molecules

In another aspect, the present invention features multispecificmolecules comprising an anti-IFN-α antibody, or an antigen-fragmentthereof, of the invention. Such multispecific molecules includebispecific molecules comprising at least one first binding specificityfor IFN-α and a second binding specificity for a second target epitope.

One type of bispecific molecules are bispecific antibodies. Bispecificantibodies are antibodies that have binding specificities for at leasttwo different epitopes. Methods for making bispecific antibodies areknown in the art, and traditional production of full-length bispecificantibodies is usually based on the coexpression of two immunoglobulinheavy-chainlight-chain pairs, where the two chains have differentspecificities (Millstein et al., Nature, 305: 537-539 (1983)).Bispecific antibodies can be prepared as full-length antibodies orantibody fragments (e.g. F(ab′)2 bispecific antibodies) or any otherantigen-binding fragments described herein.

Other multispecific molecules include those produced from the fusion ofa IFN-α-binding antibody moiety to one or more other non-antibodyproteins. Such multispecific proteins and how to construct them havebeen described in the art. See, e.g., Dreier et al. (Bioconjug. Chem.9(4): 482-489 (1998)); U.S. Pat. No. 6,046,310; U.S. Patent PublicationNo. 20030103984; European Patent Application 1 413 316; US PatentPublication No. 20040038339; von Strandmann et al., Blood (2006;107:1955-1962.), and WO 2004056873.

Multispecific molecules with more than two valencies are alsocontemplated. For example, trispecific antibodies can be prepared. Tuttet al., J. Immunol, 147: 60 (1991).

The multispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities using methods known inthe art. For example, each binding specificity of the multispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-5-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)2 or ligand x Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described or reviewed in, for example in U.S.Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513;5,476,786; 5,013,653; 5,258,498; 5,482,858; U.S. Patent applicationpublication 20030078385, Kontermann et al., (2005) Acta PharmacologicalSinica 26(1):1-9; Kostelny et al., (1992) J. Immunol. 148(5):1547-1553;Hollinger et al., (1993) PNAS (USA) 90:6444-6448; and Gruber et al.(1994) J. Immunol. 152: 5368.

Antibody Derivatives

Antibody derivatives (or immunoconjugates) within the scope of thisinvention include anti-IFN-α antibodies conjugated or covalently boundto a second agent.

For example, in one aspect, the invention provides immunoconjugatescomprising an antibody conjugated or covalently bonded to a cytotoxicagent, which cytotoxic agent can be selected from therapeuticradioisotopes, toxic proteins, toxic small molecules, such as drugs,toxins, immunomodulators, hormones, hormone antagonists, enzymes,oligonucleotides, enzyme inhibitors, therapeutic radionuclides,angiogenesis inhibitors, chemotherapeutic drugs, vinca alkaloids,anthracyclines, epidophyllotoxins, taxanes, antimetabolites, alkylatingagents, antibiotics, COX-2 inhibitors, SN-38, antimitotics,antiangiogenic and apoptotoic agents, particularly doxorubicin,methotrexate, taxol, CPT-11, camptothecans, nitrogen mustards,gemcitabine, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, pyrimidine analogs, purine analogs, platinum coordinationcomplexes, Pseudomonas exotoxin, ricin, abrin, 5-fluorouridine,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin and others (see, e.g., Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995); Goodman and Gilman's ThePharmacological Basis of Therapeutics (McGraw Hill, 2001); Pastan et al.(1986) Cell 47:641; Goldenberg (1994) Cancer Journal for Clinicians44:43; U.S. Pat. No. 6,077,499; the entire disclosures of which areherein incorporated by reference). It will be appreciated that a toxincan be of animal, plant, fungal, or microbial origin, or can be createdde novo by chemical synthesis.

In another embodiment, the antibody is derivatized with a radioactiveisotope, such as a therapeutic radionuclide or a radionuclide suitablefor detection purposes. Any of a number of suitable radioactive isotopescan be used, including, but not limited to, 1-131, Indium-111,Lutetium-171, Bismuth-212, Bismuth-213, Astatine-211, Copper-62,Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32,Phosphorus-33, Scandium-47, Silver-111, Gallium-67, Praseodymium-142,Samarium-153, Terbium-161, Dysprosium-166, Holmium-166, Rhenium-186,Rhenium-188, Rhenium-189, Lead-212, Radium-223, Actinium-225, Iron-59,Selenium-75, Arsenic-77, Strontium-89, Molybdenum-99, Rhodium-105,Palladium-109, Praseodymium-143, Promethium-149, Erbium-169,Iridium-194, Gold-198, Gold-199, and Lead-211. In general, theradionuclide preferably has a decay energy in the range of 20 to 6,000keV, preferably in the ranges 60 to 200 keV for an Auger emitter,100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alphaemitter. Also preferred are radionuclides that substantially decay withgeneration of alpha-particles.

The antibody conjugates of the invention can be used to modify a givenbiological response, where the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-y; or, biological response modifiers suchas, for example, lymphokines, interleukin-I (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

The second agent can be linked to the antibody directly or indirectly,using any of a large number of available methods. For example, an agentcan be attached at the hinge region of the reduced antibody componentvia disulfide bond formation, using cross-linkers such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP), or via a carbohydrate moiety inthe Fc region of the antibody (see, e.g., Yu et al. (1994) Int. J.Cancer 56: 244; Wong, Chemistry of Protein Conjugation and Cross-linking(CRC Press 1991); Upeslacis et al., “Modification of Antibodies byChemical Methods,” in Monoclonal antibodies: principles andapplications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal antibodies: Production,engineering and clinical application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995), Cattel et al. (1989) Chemistry today7:51-58, Delprino et al. (1993) J. Pharm. Sci 82:699-704; Arpicco et al.(1997) Bioconjugate Chemistry 8:3; Reisfeld et al. (1989) Antibody,Immunicon. Radiopharm. 2:217; the entire disclosures of each of whichare herein incorporated by reference). See, also, e.g. Amon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.,(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

In other embodiments, the second agent is a detectable moiety, which canbe any molecule that can be quantitatively or qualitatively observed ormeasured. Examples of detectable markers useful in the conjugatedantibodies of this invention are radioisotopes, fluorescent dyes, or amember of a complementary binding pair, such as a member of any one of:and antigen/antibody (other than an antibody to IFN-α),lectin/carbohydrate; avidin/biotin; receptor/ligand; or molecularlyimprinted polymer/print molecule systems.

The second agent may also or alternatively be a polymer, intended to,e.g., increase the circulating half-life of the antibody. Exemplarypolymers and methods to attach such polymers to peptides are illustratedin, e.g., U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.Additional illustrative polymers include polyoxyethylated polyols andpolyethylene glycol (PEG) moieties. As used herein, the term“polyethylene glycol” is intended to encompass any of the forms of PEGthat have been used to derivatize other proteins, such as mono (C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.For example, a full-length antibody or antibody fragment can beconjugated to one or more PEG molecules with a molecular weight ofbetween about 1,000 and about 40,000, such as between about 2000 andabout 20,000, e.g., about 3,000-12,000. To pegylate an antibody orfragment thereof, the antibody or fragment typically is reacted withpolyethylene glycol (PEG), such as a reactive ester or aldehydederivative of PEG, under conditions in which one or more PEG groupsbecome attached to the antibody or antibody fragment. Preferably, thepegylation is carried out via an acylation reaction or an alkylationreaction with a reactive PEG molecule (or an analogous reactivewater-soluble polymer). In certain embodiments, the antibody to bepegylated is an aglycosylated antibody. Methods for pegylating proteinsare known in the art and can be applied to the antibodies of theinvention. See for example, EP 0 154 316 by Nishimura et al. and EP 0401 384 by Ishikawa et al.

Antibody Characterization

After production or purification, or as part of a screening or selectionprocedure, the functional characteristics of an anti-IFN-α antibody ofthe invention can be investigated.

The following are brief descriptions of exemplary assays for antibodycharacterization. Some are further described in other sections and/ordescribed in the Examples.

Binding Assays

The present invention provides for antibodies, and antigen-bindingfragments and immunoconjugates thereof, that bind IFN-α. Any of a widevariety of assays can be used to assess binding of an antibody to IFN-α.Protocols based upon ELISAs, radioimmunoassays, Western blotting,BIACORE, and competition assays, inter alia, are suitable for use andare well known in the art.

For example, simple binding assays can be used, in which a test antibodyis incubated in the presence of a target protein or epitope (e.g., anIFN-α protein subtype selected from A, 2, B2, C, F, G, H2, I, J1, K, 4a,4b, WA, 1 and D, a portion thereof, or a combination of any thereof),unbound antibodies are washed off, and the presence of bound antibodiesis assessed using, e.g., RIA, ELISA, etc. Such methods are well known tothose of skill in the art. Any amount of binding above the amount seenwith a control, non-specific antibody indicates that the antibody bindsspecifically to the target.

In such assays, the ability of the test antibody to bind to human IFN-αcan be compared with the ability of a (negative) control protein, e.g.an antibody raised against a structurally unrelated antigen, or a non-Igpeptide or protein, to bind to the same target. Antibodies or fragmentsthat bind to IFN-α using any suitable assay with 25%, 50%, 100%, 200%,1000%, or higher increased affinity capacity ?relative to the controlprotein, are said to “specifically bind to” or “specifically interactwith” the target, and are preferred for use in the therapeutic methodsdescribed below. The ability of a test antibody to affect the binding ofa (positive) control antibody against IFN-α, e.g. a humanized ACO-1 orACO-2 antibody, may also be assessed.

In one aspect, the invention provides for humanized anti-IFN-αantibodies sharing biological characteristics and/or substantial VHand/or VL sequence identity with humanized ACO-1 or ACO-2 antibodies.One exemplary biological characteristic is the binding to the ACO-1 orACO-2 epitope, i.e., the respective regions in the extracellular domainof certain IFN-α protein subtypes to which the ACO-1 and ACO-2antibodies bind. To screen for antibodies that bind to the ACO-1 orACO-2 epitope, a routine cross-blocking assay, such as that described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed.

In an exemplary cross-blocking or competition assay, ACO-1 or ACO-2(control) antibody and a test antibody are admixed (or pre-adsorbed) andapplied to a sample containing IFN-α. In certain embodiments, one wouldpre-mix the control antibodies with varying amounts of the test antibody(e.g., 1:10 or 1:100) for a period of time prior to applying to theIFN-α-containing sample. In other embodiments, the control and varyingamounts of test antibody can simply be admixed during exposure to theantigen/target sample. As long as one can distinguish bound from freeantibodies (e.g., by using separation or washing techniques to eliminateunbound antibodies) and the control antibody from test antibody (e.g.,by using species- or isotype-specific secondary antibodies, byspecifically labeling the control antibody with a detectable label, orby using physical methods such as mass spectrometry to distinguishbetween different compounds) one will be able to determine if the testantibody reduces the binding of the control antibody to the antigen,indicating that the test antibody recognizes substantially the sameepitope as the control. In this assay, the binding of the (labeled)control antibody in the presence of a completely irrelevant antibody isthe control high value. The control low value is be obtained byincubating the labeled (positive) control antibody with unlabeledcontrol antibody, where competition would occur and reduce binding ofthe labeled antibody.

In a test assay, a significant reduction in labeled antibody reactivityin the presence of a test antibody is indicative of a test antibody thatrecognizes the same epitope, i.e., one that “cross-reacts” with thelabeled control antibody. Any test antibody or compound that reduces thebinding of the labeled control to the antigen/target by at least 50% ormore preferably 70%, at any ratio of control:test antibody or compoundbetween about 1:10 and about 1:100 is considered to be an antibody orcompound that binds to substantially the same epitope or determinant asthe control. Preferably, such test antibody or compound will reduce thebinding of the control to the antigen/target by at least 90%.Nevertheless, any compound or antibody that reduces the binding of acontrol antibody or compound to any measurable extent can be used in thepresent invention.

Biological Activity

Differentiation of Monocytes. The generation of activated T and Blymphocytes requires the recruitment and maturation of antigenpresenting cells (“APCs”). These APCs include B cells,monocytes/macrophages and dendritic cells. The serum of SLE patientscontains IFN-α which can activate DCs and the activated activity canblocked with humanized antibody preparations according to the invention.Methods to detect and quantitate this activity are described in thescientific and patent literature (see, e.g., paragraphs 0136 through0150 of patent publication number US20040067232A1, relevant portions ofwhich are hereby incorporated herein by reference).

Activation of the MxA promoter. The ability of IFN-α to activate the MxApromoter, and the ability of the anti-IFN-α monoclonal antibodies of theinvention to block this activation can be measured using reporter gene(RG) assays where the MxA promoter is fused to a reporter gene, such aschloramphenicol acetyltransferase (CAT) or luciferase (luc), preferablyluciferase. Assays for CAT and luciferase are known to those of skill inthe art. Preferably, the activity of the MxA promoter is measured inA549 cells stably transformed with an MxA promoter/reporter gene fusionconstruct. A549 cells are a lung carcinoma cell line available throughthe ATCC (product number CC1-185). The MxA (a.k.a. Mxl) promoter can behuman, mouse or rat. The sequence and structure of the human MxApromoter is disclosed in Genbank Accession number X55639, Chang et al.(1991) Arch Virol. 117:1-15; and Ronni et al. (1998) J InterferonCytokine Res. 18:773-781. Human MxA promoter/luciferase fusionconstructs and luciferase assays are disclosed in patent publicationUS20040209800 and Rosmorduc et al. (1999) J of Gen Virol 80:1253-1262.Human MxA promoter/CAT fusion constructs and CAT assays are disclosed inFernandez et al. (2003) J Gen Virol 84:2073-2082 and Fray et al. (2001)J Immunol Methods 249:235-244. The mouse MxA (Mxl) promoter is disclosedin Genbank accession number M21104; Hug et al. (1988) Mol Cell Biol8:3065-3079; and Lleonart et al. (1990) Biotechnology 8:1263-1267. Amouse MxA promoter/luciferase fusion construct and a luciferase assayare disclosed in Canosi et al. (1996) J Immunol Methods 199:69-67.

Cytopathic effect inhibition (CPE) assays. CPE assays are based on theantiviral activity of interferon. In general, a suitable cell line isinfected with a virus in the presence of interferon, and the inhibitoryactivity of interferon is quantified on viral propagation or replicativeprocesses. The readout of the assay may be based on reduction of virusyield, reduction of viral cytophatic effect, reduction of viral proteinof RNA synthesis, reduction of viral plaque formation. The cytopathicassay may be used to determine the neutralizing effect of antibodies onthe activity of interferon. Exemplary CPE assays are described inMeager, A. 1987. Quantification of interferons by anti-viral assays andtheir standardization. In: Clemens, M. J., Morris, A. G., Gearing, A. J.H. (Eds), Lymphokines and interferons: A Practical Approach. IRL, Press,Oxford, p. 129 and Grossberg and Sedmak, 1984. Assays of interferons In:Billiau, A. (Ed) Interferon, vol. 1: General and Applied Aspects.Elsevier, Amsterdam, p. 189, and in Example 6, paragraphs 157-164, andFIG. 1 of PCT publication WO2006086586.

Pharmaceutical Formulations

Another object of the present invention is to provide a pharmaceuticalformulation comprising a [the protein] compound which is present in aconcentration from 10-500 mg/ml, such as e.g. 20-300 mg/ml, preferably30-100 mg/ml, and most preferably 50-100 mg/ml, and wherein saidformulation has a pH from 2.0 to 10.0. The formulation may furthercomprise a buffer system, preservative(s), tonicity agent(s), chelatingagent(s), stabilizers and surfactants. In one embodiment of theinvention the pharmaceutical formulation is an aqueous formulation, i.e.formulation comprising water. Such formulation is typically a solutionor a suspension. In a further embodiment of the invention thepharmaceutical formulation is an aqueous solution. The term “aqueousformulation” is defined as a formulation comprising at least 50% w/wwater. Likewise, the term “aqueous solution” is defined as a solutioncomprising at least 50% w/w water, and the term “aqueous suspension” isdefined as a suspension comprising at least 50% w/w water.

In another embodiment the pharmaceutical formulation is a freeze-driedformulation, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In another embodiment the pharmaceutical formulation is a driedformulation (e.g. freeze-dried or spray-dried) ready for use without anyprior dissolution.

Diagnostic Applications

The IFN-α-antibodies of the invention also have non-therapeuticapplications. For example, anti-IFN-α antibodies may also be useful indiagnostic assays for IFN-α protein, e.g. detecting its expression inspecific cells, tissues, or serum. For example, anti-IFN-α antibodiescould be used in assays selecting patients for anti-IFN-α treatment. Forsuch purposes, the anti-IFN-α antibodies could be used for analyzing forthe presence of IFN-α in serum or tissue specimens. For diagnosticapplications, the antibody typically will be labeled with a detectablemoiety.

Therapeutic Applications

Methods of treating a patient using a humanized anti-IFN-α antibody asdescribed herein are also provided for by the present invention. In oneembodiment, the invention provides for the use of a humanized antibodyas described herein in the preparation of a pharmaceutical compositionfor administration to a human patient. Typically, the patient suffersfrom, or is at risk for, an autoimmune or inflammatory disease ordisorder associated with abnormal expression of at least one IFN-αsubtype selected from the group consisting of subtypes A, 2, B2, C, F,G, H2, I, J1, K, 4a, 4b, and WA.

Exemplary conditions or disorders to be treated with the antibodies ofthe invention, include, but are not limited to lupus (e.g., systemiclupus erythematosis (SLE)), rheumatoid arthritis, juvenile chronicarthritis, osteoarthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjogren's syndrome, vasculitis, systemic vasculitis,sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia,paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia(idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia),thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus,immune-mediated renal disease (glomerulonephritis, tubulointerstitialnephritis), demyelinating diseases of the central and peripheral nervoussystems such as multiple sclerosis, idiopathic demyelinatingpolyneuropathy or Guillain-Barre syndrome, and chronic inflammatorydemyelinating polyneuropathy, hepatobiliary diseases such as infectioushepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses),autoimmune chronic active hepatitis, primary biliary cirrhosis,granulomatous hepatitis, and sclerosing cholangitis, inflammatory boweldisease (ulcerative colitis, Crohn's disease), celiac disease,gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, and transplantation associated diseases including graftrejection and graft-versus-host-disease. In a specific embodiment, thedisease, condition or disorder is selected from lupus, Sjogren'ssyndrome, psoriasis, diabetes mellitus, rheumatoid arthritis, andjuvenile dermatomyotosis. In another specific embodiment, the disease,condition, or disorder is SLE. For example, in one aspect, theanti-IFN-α antibody is used in combination with one or more otheranti-inflammatory agents, including, but not limited to, analgesicagents, immunosuppressive agents, corticosteroids, and anti-TNFα agentsor other anti-cytokine or anti-cytokine receptor agents, andanti-angiogenic agents. Specific examples include metothrexate, TSG-6,Rituxan®, and CTLA4-Fc fusion proteins. Further examples of combinationtherapies are provided below.

Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. For example, the article of manufacture can comprisea container containing a humanized anti-IFN-α antibody as describedherein together with instructions directing a user to treat a disordersuch as an autoimmune or inflammatory disease or disorder in a humanwith the antibody in an effective amount. The article of manufacturetypically comprises a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition that is effective for treating the condition and may have asterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is thehumanized anti-IFN-α antibody herein, or an antigen-binding fragment orantibody derivative (e.g., an immunoconjugate) comprising such anantibody. The label or package insert indicates that the composition isused for treating the condition of choice, such as, e.g., SLE.

Moreover, the article of manufacture may comprise (a) a first containerwith a composition contained therein, wherein the composition comprisesthe human or humanized antibody herein, and (b) a second container witha composition contained therein, wherein the composition comprises atherapeutic agent other than the human or humanized antibody. Thearticle of manufacture in this embodiment of the invention may furthercomprise a package insert indicating that the first and secondcompositions can be used in combination to treat an autoimmune orinflammatory disease or disorder. Such therapeutic agents may be any ofthe adjunct therapies described in the preceding section. Alternatively,or additionally, the article of manufacture may further comprise asecond (or third) container comprising a pharmaceutically acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes.

In a first aspect, the present invention thus relates to a humanizedantibody, or an antigen-binding fragment thereof, that specificallybinds human interferon-α (IFN-α), which humanized antibody is ahumanized version of murine antibody ACO-1 or ACO-2, or of a combinationthereof, comprising fewer donor amino acid residues than the murinecomplementary determining regions (CDRs) according to Kabat.

In a second aspect, the present invention thus relates to a humanizedantibody that specifically binds IFN-α, or an antigen-binding fragmentthereof, wherein said antibody is capable of binding IFN-α subtypes A,2, B2, C, F, G, H2, I, J1, K, 4a, 4b and WA, but not subtypes 1 or D,and wherein said antibody comprises fewer donor amino acid residues thanthe non-human CDRs according to Kabat.

According to one embodiment, the CDR H2 donor residues comprise Kabatresidues 50-59. In another embodiment, said antibody competes withand/or binds to the same epitope on an IFN-α subtype as ACO-1 and/orACO-2 antibody.

According to a preferred embodiment, the antibody is an IgG4 subtype. Ina preferred embodiment, the antibody comprises a CDR H2 sequenceaccording to SEQ ID NO: 21.

In a third aspect, the present invention relates to a method forproducing an antibody according to the invention, wherein said methodcomprises incubating a host cell encoding said antibody underappropriate conditions and subsequently isolating said antibody. Theinvention furthermore relates to antibodies obtained by or obtainable bysuch methods.

In a fourth aspect, the present invention relates to a compositioncomprising an antibody according to the invention. The inventionfurthermore relates to a process for the preparation of a compositionaccording to the invention, wherein said method comprises mixingantibody or a fragment thereof with excipients. The inventionfurthermore relates to compositions obtained by or obtainable by suchmethods.

In a fifth aspect, the present invention relates to a method ofpreventing, managing, treating or ameliorating an IFN-α relatedinflammatory disease or disorder, said method comprising administeringto a subject in need thereof a prophylactically or therapeuticallyeffective amount of an antibody according to the invention.

Finally, the present invention relates to use of an antibody accordingto the invention for preparation of a medicament suitable for treatmentof an inflammatory disease.

EXAMPLES

Further details of the invention are illustrated by the followingnon-limiting Examples.

Example 1 Sequencing of Murine ACO-1 and ACO-2 Antibodies

This example describes sequencing and recombinant expression of themurine antibodies ACO-1 and ACO-2, described in WO20060086586, as wellas BLAST searches on the ACO-2 VH and VL sequences.

Antibody Cloning and Sequencing

Total RNA was extracted from hybridomas (ACO-1.5.2 and ACO-2.2.1) usingthe RNeasy® kit (#634914) from Qiagen®. cDNA was synthesized from 1 μgtotal RNA using SMART™-RACE cDNA amplification kit from Clontech®Laboratories, Inc. The reaction was run at 42° C. for 1.5 h and thesamples were diluted in 75 μl tricine-EDTA. PCR amplification of thetarget was carried out in 50 μl reactions using 5 μl of cDNA astemplate. The forward primer for both heavy and light chain wasuniversal primer mix (UPM) that was included in the SMART™ RACE kit. Thereverse primer sequence for ACO-1 heavy chain (HC) was designed asfollows:

-   -   5′-CTGGGCCAGGTGCTGGAGG (SEQ ID NO:11) and, for ACO-1 light chain        (LC)    -   5′-CTAACACTCATTCCTGTTGAAGCTC (SEQ ID NO:12).        The reverse primer sequence for ACO-2 heavy chain (HC) was        designed as follows:    -   5′-CTAGCTAGCTCATTTACCCGGAGACCGGGAGATGG (SEQ ID NO:26) and, for        ACO-2 light chain (LC): 5′-GCTCTAACACTCATTCCTGTTGAAGCTCTTG (SEQ        ID NO:27).

The PCR reactions were carried out using the Advantage® HF PCR kit fromClontech® Laboratories, Inc. and the PCR program was run with a singledenaturing step at 94° C./2 min followed by 24 cycles as given: 94°C./30 sec.; 55° C./30 sec.; 72° C./1.5 min. The final extension step was72° C./10 min. The PCR products were identified on a 1% agarose gelcontaining ethidium bromide. The PCR products were purified from the gelusing GFX™ Purification kit from GE Healthcare™ followed by cloning intoZero Blunt® TOPO® PCR Cloning Kit (#K2875-40) and transformed into TOP10E. coli cells from Invitrogen® Corp.

DNA was extracted from E. coli colonies using the miniprep kit (#27106)from Qiagen®. Plasmids were sequenced at MWG Biotech™ AG, Matinsried,Germany using the sequencing primers M13 rev (−29) and M13 uni (−21). HCand LC were verified from the identified sequences by using VectorNTI®software. All procedures based on kits were performed according tomanufacturer directions.

From the ACO-1.5.2 hybridoma cells a single kappa LC and a single IGg2aHC were cloned, having the following nucleic acid and amino acidsequences.

ACO-1 VH sequence (SEQ ID NO: 13 (signal peptide included)):atgggatggagctatatcatgctctttttggtagcaacagctacagatgtccac-tcccaggtccaactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaa-gctgtcctgtaaggcttctggctacaccttcaccaactactggatgcactgggtgaa-gcagaggcctggacaaggccttgagtggattggagagattaatcctagccacggtcgtacta-tctacaatgaaaacttcaagagcaaggccacactgactgtaga-caaatcctccatcacagccttcatgcaactcagcagcctgacatctgaggactctgcggtc-tatttctgtgcaagagggggactgggacccgcctggtttgcttactggggccaagggactctggtcactgtctctgcaACO-1 VL sequence (SEQ ID NO: 14 (signal peptide included)):atggattttcaagtgcagattttcagcttcctgctaatcagtgtctcag-tcataatgtccagaggacaaattgttctcacccagtctccag-caatcatgtctgcttctcctggggagaaggtcaccttgacctgcagtgccggctcaagtg-tagattccagctatttgtactggtaccagcagaagccaggatcctcccccaaactctggat-ttatagcacatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgg-gacctcttactctctcacaatcagcagcatggaggctgaa-gatgctgcctcttatttctgccatcagtggagtagttacccattcacgttcggctcggggacaaaattggaaataaaacggACO-1 VH (SEQ ID NO: 1 (signal peptide excluded))QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGEINPSHGRTIYNENFKSKATLTVDKSSITAFMQLSSLTSEDSAVYFCARGGLGPAWFAYWGQGTLVTVS AACO-1 VL (SEQ ID NO: 4 (signal peptide excluded))QIVLTQSPAIMSASPGEKVTLTCSAGSSVDSSYLYVVYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPFTFGSGTKLEI KR

From the ACO-2.2.1 hybridoma cells a single kappa type ACO-2 light chainand a single IGg2b ACO-2 heavy chain were cloned, with the followingnucleic acid and amino acid sequences.

ACO-2 VH sequence (SEQ ID 28 (signal peptide included))atgggatggagctatatcatcctctttttggtagcagcagctacagatgtccactcccaggtccaactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaagctgtcctgcaaggcctctggctacagcttcaccagctactggatgcactgggtgaagcagaggcctggacaaggccttgagtggattggagagattaatcctagccacggtcgtactagctacaatgagaacttcaagagcaaggccacactgactgtagacaaatcctccaacatagtctacatgcaactcagcagcctgacatctgaggactctgcggtctattactgtgtaagagggggactgggacccgcctggtttgcttactggggccaagggactctggtcactgtctctgtaACO-2 VL sequence (SEQ ID NO: 29 (signal peptide included))atggattttcaagtgcagattttcagcttcctgctaatcagtgtctcagtcataatgtccagaggacaaattgttctcacccagtctccagcaatcatgtctgcatctcctggggagaaggtcaccttgacctgcagtgccggctcaagtgtaggttccagctacttttactggtaccagcagaagccaggatcctcccccaaactctggatttatggcacatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgcctcttatttctgccatcagtggagtagttatccattcacgttcggctcggggacaaaattggaaataaaacggACO-2 VH sequence (SEQ ID NO: 7 (signal peptide excluded)):QVQLQQPGAELVKPGASVKLSCKASGYSFTSYWMHWVKQRPGQGLEWIGEINPSHGRTSYNENFKSKATLTVDKSSNIVYMQLSSLTSEDSAVYYCVRGGLGPAWFAYWGQGTLVTV SVACO-2 VL sequence (SEQ ID NO: 9 (signal peptide excluded)):QIVLTQSPAIMSASPGEKVTLTCSAGSSVGSSYFYVVYQQKPGSSPKLWIYGTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPFTFGSGTKLEIKR

The ACO-2 CDR sequences according to the Kabat definitions were found tobe as follows.

(SEQ ID NO: 22) CDR-H1: SYWMH (SEQ ID NO: 23) CDR-H2: EINPSHGRTSYNENFKS(SEQ ID NO: 17) CDR-H3: GGLGPAWFAY (SEQ ID NO: 24) CDR-L1: SAGSSVGSSYFY(SEQ ID NO: 25) CDR-L2: GTSNLAS (SEQ ID NO: 20) CDR-L3: HQWSSYPFT

Example 2 Design of Humanized ACO-1 and Identification of PotentialBack-Mutation Residues

Identification and Characterization of Mouse ACO-1 CDRs

The ACO-1 CDR sequences according to the Kabat definitions were found tobe as follows.

(SEQ ID NO: 15) CDR-H1: NYWMH (SEQ ID NO: 16) CDR-H2: EINPSHGRTIYNENFKS(SEQ ID NO: 17) CDR-H3: GGLGPAWFAY (SEQ ID NO: 18) CDR-L1: SAGSSVDSSYLY(SEQ ID NO: 19) CDR-L2: STSNLAS (SEQ ID NO: 20) CDR-L3: HQWSSYPFT

Identification of Human Germline

A 3D protein structure model was built using MOE (Molecular OperatingEnvironment; available at www.chemcomp.com) with a structural templatefrom the Protein Database Bank (PDB): 1Z3G. The PDB is described inBerman et al. (Nucl Acids Res 2000; 28:235-242), and is available atwww.rcsb.org/pdb. Based on an analysis of antibody-antigen complexes inthe PDB database, the most probable residues in the paratope were foundto be residues 23-35, 49-58, 93-102 of the ACO-1 VH, and 24-34, 49-56,89-97 of the ACO-1 VL. Using MOE, residues interacting (hydrophobic,hydrogen binding, charge interaction) with the paratope were identifiedand the combined set of residues (paratope+interacting residues) weretaken as the so-called mask of ACO-1 shown in FIG. 1.

Searching the germline V databases with the ACO-1 VH and ACO-1 VLreturned the following potential framework templates (E-value given inparenthesis):

-   -   VH: VH1_(—)46 (3e-038) VH1_f (6e-037), VH1_(—)02 (6e-037),        VH1_(—)03 (1e-036), VH1_(—)24 (2e-034),    -   VL: VKIII_L6 (9e-035), VKIII_A11 (4e-034), VKIII_A27 (8e-034),        VKIII_L25 (1e-033), VKI_L8 (1e-033).    -   Searching the germline databases with the mask returned the        following potential framework templates (E-value given in        parenthesis):    -   VH: VH1_(—)46 (3e-011) VH1_(—)02 (6e-011), VH1_f (1e-010), VH5_a        (4e-010), VH1_(—)03 (4e-010),    -   VL: VKIII_A11 (5e-009), VKIII_L6 (7e-009), VKIII_A27 (9e-009),        VKIII_L25 (3e-008), VKI_L9 (6e-008).

After manual inspections of the alignments and the hits, VH1_(—)46 andVKIII_L6 were selected as the human scaffolds. Other templates could bechosen to alter or optimize, e.g., the physical-chemical properties ofthe humanized antibody. JH4 and JK2 were selected as germline J-segments(SEQ ID NO:2 and 5, respectively).

Design of Optimal Humanized ACO-1

Humanization was performed with the following rules:

-   -   Residues outside the mask were taken as human.    -   Residues inside the mask and inside the Kabat CDR were taken as        murine.    -   Residues inside the mask and outside the Kabat CDR with        mouse/germline consensus were taken as the consensus sequence.    -   Residues inside the mask and outside the Kabat CDR with        mouse/germline were taken as the germline sequence, but the        murine difference were subject to potential back mutations.

The CDRs of the optimal hzACO-1 antibody obtained were (according to theKabat definitions):

(SEQ ID NO: 15) CDR_H1 NYWMH (SEQ ID NO: 21) CDR_H2 EINPSHGRTIYAQKFQG(SEQ ID NO: 17) CDR_H3 GGLGPAWFAY (SEQ ID NO: 18) CDR_L1 SAGSSVDSSYLY(SEQ ID NO: 19) CDR L2 STSNLAS (SEQ ID NO: 20) CDR_L3 HQWSSYPFT

Using the above humanization method, designing a mask of residuespredicted to constitute the paratope based on a 3D model of hzACO-1 andIFN-αA, in contrast to simple CDR grafting, a hzACO-1 antibody withfewer murine residues was obtained, since the peptide comprising the 5C-terminal amino acids of the optimized hzACO-1 CDR H2 sequence(highlighted in bold above) was identical to the corresponding humanframework sequence, while the corresponding peptide in the ACO-1 CDR H2sequence according to the Kabat definitions was of murine origin.Additionally, the CDR H1 sequence for a humanized ACO-1 antibodyidentified in the present analysis was shorter than the one described inWO2006/086586. The optimized hzACO-1 antibody, or an antibody orantigen-binding fragment comprising at least a portion of the hzACO-1 VHsequence, can thus provide for a reduced risk for ahuman-anti-mouse-antibody (HAMA)-response in a human patient.

In addition, the replacement of the sequence AQK instead of NEN inposition 60-62 in heavy chain has the advantage of avoiding twoasparagine residues which may be prone to deamidation.

Identification of Potential Backmutations.

The analysis of ACO-1 VH and VL sequences is illustrated in FIG. 1. InFIG. 1, the resulting humanized ACO-1 (hzACO-1) VH (SEQ ID NO:3) and VL(SEQ ID NO:6) sequences are shown with potential back-mutation residuesas human, i.e., without any back-mutations. The following back-mutationvariants in the framework regions were identified for obtaining one ormore optimized hzACO-1 antibodies, which are often required in order toretain the affinity of the original mouse antibody:

-   -   hzACO-1 VH: wild-type (i.e., no back-mutation), V5Q, M69L, R71V,        T73K, S761, V78A and any combination any thereof;    -   hzACO-1 VL: wild-type, E1Q, L47W, I58V, F71Y and any combination        of any thereof;    -   in various heavy-light chain combinations.

Example 3 Design of ACO-2-Based Variants of hzACO-1 for AffinityMaturation of hzACO-1

As shown in FIG. 2, amino acid sequence alignments of the ACO-1 andACO-2 VH and VL sequences revealed a high sequence identity between therespective light and heavy chains. Without being limited to theory, itis possible that the antibodies derived from the same precursor cell asthey had the same V-D-J rearrangement and contained 3 identicalmutations compared to the germline sequence. In addition, the antibodiesdiffered at 13 amino acid residues, possibly due to subsequent somatichyper-mutations.

Out of the 13 non-identical amino acid residues in the ACO-1 and ACO-2VH and VL domains, 9 residues were selected for mutational analysis inorder to improve the affinity of the humanized ACO-1 antibody (FIG. 3).Single additions of ACO-2 derived hypermutations could potentiallyidentify deleterious and beneficial amino acid residues and by allowingthe introduction of only the beneficial amino acids improve the affinitybeyond the original mouse ACO-1 and ACO-2 antibodies. The targetedresidues were chosen based on their position within one of the light orheavy chain CDRs (according to the Kabat definition) or based on theirlocation within regions outlined as potential antigen-interactingregions based on antigen-antibody 3D-modelling.

The following variants were identified for obtaining one or moreoptimized hzACO-1 antibodies:

-   -   hzACO-1 VH: wild-type, T28S, N31S, I58S, S76N, T77I and A93V,        and any combination of at least two mutations selected from        T28S, N31 S, I58S, S76N, T77I and A93V;    -   hzACO-1 VL: wild-type, D29G, L33F, S50G and any combination of        at least two mutations selected from D29G, L33F, and S50G,    -   in various heavy-light chain combinations.

Residues were mutated separately from ACO-1 sequence to ACO-2 sequencewithin the hzACO-1 light and heavy chain constructs, in order toevaluate the individual contribution of each residue to antigen binding.A series of combination mutants was also generated.

Example 4 Cloning of ACO-1, ACO-2, hzACO-1 and Site-directed Mutagnesis

ACO-1, ACO-2, and hzACO-1

The VH and VL sequences were transferred to CMV promoter-basedexpression vectors (pTT vectors) suitable for transient expression inthe HEK293-EBNA (HEK293-6E) expression system described by Durocher etal. (Nucleic Acids Res. 2002; 30(2):E9). In addition to theCMV-promoter, the vectors contain a pMB1 origin, an EBV origin and theAmpR gene. The VH of ACO-1 and ACO-2 were cloned into the CMV-basedvector containing the constant region for mouse IgG2a and IgG2b,respectively. The full length LC was transferred to the empty CMV-basedvector for both ACO-1 and ACO-2. The DNA sequences for the variableregions of hzACO-1 were ordered from Geneart, Regensburg, Germany. Thedelivered sequence for the hzACO-1 VH was transferred to the expressionvector containing the constant region for human IgG4(S241P) (containinga S241P mutation in the hinge region). The delivered hzACO-1 VL sequencewas transferred to the vector containing the constant region for a humankappa light chain.

Generation of Back-mutation Variants of hzACO-1

The 10 potential back-mutations identified in Example 2 were introducedseparately to hzACO-1 HC and LC constructs in order to gauge theindividual contribution of each residue to antigen binding. A fewcombinations were included as well. A variant of hzACO-1 with anextended CDR H2 (hzACO-1-Kabat CDRH2), equivalent to the Kabatdefinition of the murine CDR H2 (SEQ ID NO:16) was also generated bysite-directed mutagenesis.

Site-directed mutagenesis was performed on the hzACO-1 LC and HCexpression vectors. To generate single mutants the QuickChange®Site-Directed Mutagenesis kit from Stratagene® Corp. (cat.#200518) wasused according to the manufactures protocol. Introduction of desiredmutations was verified by sequencing plasmid DNA preparations (MWGBiotech, Matinsried, Germany) for each mutant.

The mutated LC constructs were combined with the hzACO-1 HC forexpression, and mutated HC constructs were combined with the hzACO-1 LCfor antibody expression.

-   -   Light Chain Variants with Back-Mutations:        -   hzACO-1-E1Q        -   hzACO-1-L47W        -   hzACO-1-I58V        -   hzACO-1-F71Y        -   hzACO-1-L47W,I58V    -   Heavy Chain Variants with Back-Mutations:        -   hzACO-1-V5Q        -   hzACO-1-M69L        -   hzACO-1-R71V        -   hzACO-1-T73K        -   hzACO-1-S761        -   hzACO-1-V78A        -   hzACO-1-R71V,T73K        -   hzACO-1-M69L, R71V, T73K, S761, V78A        -   hzACO-1-Kabat CDRH2

Generation of ACO-2-based Variants of hzACO-1

Site-directed mutagenesis introducing ACO-2 specific residues into thehzACO-1 antibody in order to improve affinity, as described in example3, was performed on the hzACO-1 LC and HC expression vectors. Togenerate single mutants the QuickChange® Site-Directed Mutagenesis kitfrom Stratagene® Corp. (cat.#200518) was used according to themanufactures protocol. Combination mutants were generated using both theQuickChange® Site-Directed Mutagenesis and QuickChange® MultiSite-Directed Mutagenesis kits according to the manufactures protocols(cat.#200513).

Introduction of desired mutations was verified by sequencing plasmid DNApreparations (MWG Biotech, Matinsried, Germany) for each mutant.

The mutated light chain constructs were combined with the hzACO-1 heavychain for expression and mutated heavy chain constructs were combinedwith the hzACO-1 light chain constructs for antibody expression.

-   -   Light Chain Variants with ACO-2-Derived Mutations:        -   hzACO-1-D29G        -   hzACO-1-L33F        -   hzACO-1-550G        -   hzACO-1-D29G, L33F, S50G    -   Heavy Chain Variants with ACO-2-Derived Mutations:        -   hzACO-1-T28S        -   hzACO-1-N31S        -   hzACO-1-I58S        -   hzACO-1-S76N        -   hzACO-1-T77I        -   hzACO-1-A93V        -   hzACO-1-N31S, I58S        -   hzACO-1-T28S, N31S, I58S, A93V        -   hzACO-1-S76N, T77I        -   hzACO-1-T28S, N31S, I58S, S76N, T77I, A93V        -   hzACO-1-T28S, A93V        -   hzACO-1-N31S, A93V        -   hzACO-1-T28S, N31S, A93V        -   hzACO-1-T28S, N31S

Example 5 Expression of Recombinant ACO Derived Antibodies

The ACO-1, ACO-2, hzACO-1, and hzACO-1 variants were expressed intransiently transfected HEK293 cells following a generic antibodyexpression protocol. The following describes the transfection protocolfor suspension adapted HEK293 cells. Cell maintenance: Suspensionadapted HEK293 cells were grown in GIBCO® FreeStyle™ 293 Expressionmedium (Invitrogen® Corp. cat. #: 12338-026) supplemented with 25 μg/mlGeneticin® antibiotic (Invitrogen® Corp. cat. #: 10131-019), 0.1% v/vPluronic® F-68 (Invitrogen® Corp. cat. #: 12347-019) surfactant & 1% v/vPenicillin-Streptomycin (Optional) (Invitrogen® Corp. cat. #15140-122).Cells were maintained in Erlenmeyer shaker flasks at cell densitiesbetween 0.2−2×10⁶ cells/ml in an incubator shaker at 37° C., 8% CO₂ and125 rpm.

DNA Transfection: The cell density of cultures used for transfection was0.8−1.5×10⁶ cells/ml. 0.5 μg light chain vector DNA+0.5 μg heavy chainvector DNA were used per ml cell culture. 293Fectin™ reagent(Invitrogen® Corp. cat. #: 12347-019) was used as transfection reagentat a concentration of 1 μl reagent per μg transfected DNA. The293Fectin™ reagent was diluted in 30× vol. Opti-MEM® medium (Invitrogen®Corp. cat. #: 51985-034), mixed and left at room temperature (23-25° C.)for 5 min. The DNA was diluted in 30 μl Opti-MEM® medium per μg totalDNA, mixed and left at room temperature (23-25° C.) for 5 min. The DNAand transfection reagent dilutions were mixed 1:1 and left at roomtemperature (23-25° C.) for 25 min. The DNA-293Fectin™ mix was addeddirectly to the cell culture. The transfection cell culture wastransferred to an incubator shaker at 37° C., 8% CO₂ and 125 rpm. After4-7 days, cell culture supernatants were harvested by centrifugationfollowed by filtration through a 0.22 μm PES filter (Corning® Inc. cat.#: 431098). The antibodies were analyzed as supernatants or purifiedusing standard protein A purification techniques.

Expression Level Comparison of hzACO-1 and hzACO-1-Kabat CDRH2

Transient expression levels in HEK293-6E cells were compared for hzACO-1and hzACO-1-Kabat CDRH2 to determine if the distal CDR H2 residues hadany effect on the ability of the cells to expressed either of the twoantibody variants.

HEK293-6E cells were transfected as described above with pTT-basedexpression vectors for hzACO-1 light chain and hzACO-1 or hzACO-1-KabatCDRH2 heavy chains. The transfections was performed in triplicates. Foreach antibody variant, three cultures (25 ml) were transfected using aDNA-293Fectin master mix to minimize the influence from pipettinginaccuracy. The transfected cultures were incubated in a shakerincubator for 4 days as described above. At day 4, samples wereextracted from the cultures for cell viability and cell densitymeasurements and the remaining cell culture supernatants were harvestedby centrifugation. Quantitation analysis of antibody production wasperformed by Biolayer Interferometry directly on clarified cell culturesupernatants using the FortíeBio® Octet® system and protein Abiosensors. Cell culture densities and viabilities were measured using aCedex® HiRes™ automated cell culture analyzer. Results are show in Table4 below.

TABLE 4 Expression analysis Standard Standard deviation Viable Celldeviation Cell Standard Expression (Expression Density (Viable Cellviability deviation Yield (mg/L) Yield) (×10E5 cell/ml) Density) (%)(Cell viability) hzACO-1 41 0.9 19 0.2 65 2.1 hzACO-1-Kabat CDRH2 22 0.718 0.5 65 3.2

The results in Table 4 unexpectedly show a significant difference in thetransient expression levels for hzACO-1 (humanized IFN-alpha antibodyaccording to the present invention) compared with hzACO-1-Kabat CDRH2(humanized IFN-alpha antibody humanized using traditional procedures andhence full length Kabat sequences). No difference in cell viability ordensity was observed for the cell cultures transfected with either ofthe two hzACO-1 variants. The expression level for the hzACO-1 wasapproximately 2-fold higher compared to the expression level forhzACO-1-Kabat CDRH2 antibody variant

By grafting a shorter version of CDR H2 compared to the Kabat-definedCDRH2, expression levels of the antibody were surprisingly significantlyincreased.

Without being bound by theory, it may hypothesized that the humangermlinederived residues in the hzACO-1 as compared to the hzACO-1-KabatCDRH2 antibody variant, carrying the extended CDR H2 (SEQ ID:16) affectHC folding and potentially LC-HC interaction, results in an improvedprotein stability and thus expression yield.

Such an improved level of protein expression resulting from an improvedprotein stability observed in the transient expression levels will bereflected in stable CHObased production cell lines. Therefore, bygrafting the short version of CDRH2 (SEQ ID:21) generation of ahigh-producing stable production cell line is thus possible.

Expression Level Comparison of IgG4, 1 and 2 Variants of hzACO-1

Transient expression levels in HEK293-6E cells were compared for thelead IgG4(S241P) variant of hzACO-1 and hzACO-1(IgG1) and hzACO-1(IgG2)to determine if heavy chain subclass switching had any effect onantibody expression levels.

The experiment was performed similarly to the expression assay describedabove, but with the following changes: The experiment was performed induplicates in 5 ml cultures. The cultures were incubated in filtercapped 50 ml falcon tubes in a shaker incubator at 37° C., 8% CO₂ and250 rpm.

Unpredictably, the average expression levels for hzACO-1(IgG1) andhzACO-1(IgG2) were approximately 65% of the expression level for hzACO-1(IgG4). Based on the observed reduction in protein expression (˜35%) weconclude that the combination of hzACO-1 variable domains and IgG4constant domain is superior to combinations with other chain subclassesand the development of this molecule greatly facilitate generation of ahigh-producing stable production cell line.

Example 6 Crystal Structure of IFN-α8 in Complex with hzACO-1-Fab

The crystal structure of IFN-α8 in complex with the Fab fragment ofhzACO-1 was determined and refined to 3.3 Å resolution using X-raycrystallography (FIG. 8).

Materials and Methods

IFN-α8 (amino acid 1-166 of SEQ ID NO: 30, and hzACO-1 Fab (with thelight chain sequence of SEQ ID NO: 32 and heavy chain sequence ofresidues 1-221 of SEQ ID NO: 31) were mixed, with a slight excess ofIFN-α8, and the complex was purified on a gel-filtration column. Theprotein complex hzACO-1-Fab/IFN-α8 was put in a buffer of 25 mM HEPESbuffer, pH 7.5, +25 mM NaCl and concentrated to 5 mg/ml. The complex wascrystallized in 100 mM HEPES buffer, pH 7.5, 15% PEG 10,000 and 15%Ethylene glycol, the precipitant solution. Prior to diffraction datacollection, the crystal was flash frozen in liquid N₂. The crystal wasfirst transferred to a cryo solution which was a mix of 25% v/v 99%glycerol and 75% of the precipitant solution. Diffraction data werecollected at beamline BLI911-3, MAX-Lab, Lund, Sweden. Diffraction datawere indexed and integrated using the XDS program package (Kabsch, J.Appl. Crystallogr. 1993; 26:795-800).

The three-dimensional structure was determined using the MolecularReplacement (MR) method using the PHASER program (Read, 2001, ActaCrystallogr. Sect. D-Biol. Crystallogr. 57, 1373-1382) of the CCP4package (Baily, 1994, Acta Crystallogr. Sect. D-Biol. Crystallogr. 50,760-763). The crystal structure of the hzACO-1 Fab, un-complexed, wasearlier been determined to 1.52 Å resolution (R- and R-free 0.18 and0.21, respectively), data not shown. Those 3D coordinates weresubsequently used in the MR calculations for the hzACO-1/IFN-α8 complex.The search models were divided into three parts: 1) the variable domainof hzACO-1 Fab), 2) the constant domain of the hzACO-1 Fab and 3) thePDB deposited IFNtau model, Protein Data Bank (Berman et al., 2000,Nucleic Acids Res. 28, 235-242) accession code 1B5L (RADHAKRISHNAN etal, 1999, J. MOL. BIOL. v. 286 pp. 151), mutated by the COOT program toobtain the sequence of IFN-α8, SEQ ID NO: 30. The final space groupdetermination was made by the PHASER program. The highest scores wereobtained for space group P4₁ with rotation function peaks, Rz's, of10.7, 4.4 and 2.6 σ, respectively, translation peaks, TZ's, of 24.1,26.4 and 8.0 σ, respectively, log-likelyhod gains, LLG's, of 383, 918and 1134, respectively, and with no overlaps to symmetry relatedmolecules.

Molecular replacement was followed by some adjustments to the model inthe COOT molecular graphics program after which torsional simulatedannealing up to 2000 K was applied twice, without refinement ofindividual temperature factors. Original R- and R-free values were 0.416and 0.439, respectively, and final values after simulated annealing were0.314 and 0.427, respectively. The model was then subject to manualintervention in the COOT program followed refinements using the REFMAC5program (Murshudov et al., 1997, Acta Crystallogr.

Sect. D-Biol. Crystallogr. 53, 240-255) resulting in R- and R-freevalues of 0.216 and 0.348, respectively. The model comprised residues1-21, while residues 22-23 were refined as Ala residues, 28-101 and114-164 of IFN-α8, 1-215 of the hzACO-1 light chain and 1-219 of thehzACO-1 heavy chain.

The relatively large difference between R- and R-free seen in therefinement are due to the limited resolution of the data, 3.3 Å, andthat there are substantial stretches of the IFN-α8 X-ray model that arecompletely missing in the interpretation of the electron density map.The electron density maps clearly define the residues in the stabilisedinterface of IFN-α8 to hzACO-1-Fab, while details of the IFN-α8 X-raystructure model away from the antibody site are less well defined andtherefore less accurately determined.

Results

The contacts were identified by the CONTACT computer program of the CCP4suite using a cut-off distance of 4.0 Å between the Fab and IFN-α8molecules. The resulting epitope for human hzACO-1 was found to comprisethe following residues of IFN-α8 (SEQ ID NO: 30): Ser 55, His 58, Glu59, Gln 62, Gln 63, Asn 66, Glu 97, Leu 118, Arg 121, Lys 122, Phe 124,Gln 125, Arg 126, Thr 128, Leu 129, Thr 132). Residues of hzACO-1involved in interactions with IFN-α8, the paratope, included Ser 32, Tyr33, Tyr 35, Tyr 50, Ser 51, Trp 92, Ser 93, Tyr 95 and Phe 97 of thehzACO-1 light (L) chain, (Numbering according to SEQ ID NO: 32, notKabat, and Thr 30, Asn 31, Tyr 32, Trp 33, His 35, Glu 50, Asn 52, Ser54, His 55, Arg 57, Leu 101, Gly 102, Trp 105 of the heavy (H) chain,Table 9 (Numbering according to SEQ ID NO: 31, not Kabat).

>IFN_a8 SEQ ID NO: 30CDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFEFPQEEFDDKQFQKAQAISVLHEMIQQTFNLFSTKDSSAALDETLLDEFYIELDQQLNDLESCVMQEVGVIESPLMYEDSILAVRKYFQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSINLQKRLKSKE hzACO-1 LC SEQ ID NO: 32  1 EIVLTQSPAT LSLSPGERAT LSCSAGSSVD SSYLYWYQQK PGQAPRLLIY  51STSNLASGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCH QWSSYPFTFG 101QGTKLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK 151VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ 201GLSSPVTKSF NRGEC hzACO-1Fab HC SEQ ID NO: 31   1QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGE  51INPSHGRTIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGG 101LGPAWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD 151YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY 201TCNVDHKPSNTKVDKRVESK

As can be seen in FIG. 9 the hzACO-1 interaction epitope on IFN-α8overlaps, partly, the IFNAR1 binding epitope while the IFNAR2 bindingepitope is distant from the hzACO-1 binding epitope. That suggests thatthe neutralization of IFN-α by hzACO-1 occurs by neutralization of IFN-αbinding to IFNAR1, but not IFNAR2. Accordingly, it may be envisionedthat hzACO-1 binds the IFN-α/IFNAR2 complex but inhibits formation ofthe ternary receptor complex IFN-α/IFNAR1/IFNAR2 responsible forintracellular signaling.

TABLE 5 Parameters of the IFN-α8: hzACO-1-Fab complex crystal used forstructure determination Space Group: P4₁ Cell parameters [Å]: a b C112.74 112.74 60.55 Molecular complexes/ 1  asymmetric unit:

TABLE 6 X-ray data statistics from the program XSCALE of the XDS packageNUMBER OF REFLECTIONS RESO- COM- LUTION PLETE- R-FACTOR S_norm/ [Å]OBSERVED UNIQUE POSSIBLE NESS observed expected COMPARED I/SIGMA R-Rmrgd-F S_ano 10.00  984 353 453 77.9% 4.4% 4.9% 984 18.84 5.4% 4.1% −1%6.00 4237 1461 1562 93.5% 6.8% 7.2% 4237 12.45 8.2% 7.8% 1% 5.00 39221343 1411 95.2% 9.7% 10.1% 3922 9.83 11.7% 11.1% 3% 4.00 8747 3063 318296.3% 11.6% 11.9% 8747 8.59 14.0% 13.7% 8% 3.50 8588 3106 3212 96.7%24.8% 24.9% 8588 4.43 30.3% 31.3% 9% 3.45 1102 397 410 96.8% 36.9% 37.8%1102 3.17 45.0% 44.4% 7% 3.40 1198 448 464 96.6% 41.8% 41.3% 1198 2.8252.3% 48.7% −12% 3.35 1271 468 495 94.5% 56.0% 51.5% 1271 2.33 69.9%66.9% 1% 3.30 1317 486 502 96.8% 59.6% 51.2% 1317 2.14 74.9% 67.4% −8%total 31366 11125 11691 95.2% 14.1% 14.2% 31366 7.44 17.2% 20.2% 6%

TABLE 7 Statistics from the last refinement cycle of IFN-α8: hzACO-1-Fabof the REFMAC program. DATA USED IN REFINEMENT. RESOLUTION RANGE HIGH(ANGSTROMS) 3.30 RESOLUTION RANGE LOW (ANGSTROMS) 20.23 DATA CUTOFF(SIGMA(F)) NONE COMPLETENESS FOR RANGE (%) 95.66 NUMBER OF REFLECTIONS10571 FIT TO DATA USED IN REFINEMENT. CROSS-VALIDATION METHOD THROUGHOUTFREE R VALUE TEST SET SELECTION RANDOM R VALUE (WORKING + TEST SET)0.22272 R VALUE (WORKING SET) 0.21646 FREE R VALUE 0.34845 FREE R VALUETEST SET SIZE (%) 5.0 FREE R VALUE TEST SET COUNT 552 FIT IN THE HIGHESTRESOLUTION BIN. TOTAL NUMBER OF BINS USED 20 BIN RESOLUTION RANGE HIGH3.300 BIN RESOLUTION RANGE LOW 3.384 REFLECTION IN BIN (WORKING SET) 761BIN COMPLETENESS (WORKING + TEST) (%) 96.06 BIN R VALUE (WORKING SET)0.290 BIN FREE R VALUE SET COUNT 44 BIN FREE R VALUE 0.446 B VALUES.FROM WILSON PLOT (A**2) NULL MEAN B VALUE (OVERALL, A**2) 52.314 OVERALLANISOTROPIC B VALUE. B11 (A**2) −0.96 B22 (A**2) −0.96 B33 (A**2) 1.92B12 (A**2) 0.00 B13 (A**2) 0.00 B23 (A**2) 0.00 ESTIMATED OVERALLCOORDINATE ERROR. ESU BASED ON R VALUE (A) NULL ESU BASED ON FREE RVALUE (A) 0.776 ESU BASED ON MAXIMUM LIKELIHOOD (A) 0.574 ESU FOR BVALUES BASED ON MAXIMUM 33.433 LIKELIHOOD (A**2) CORRELATIONCOEFFICIENTS. CORRELATION COEFFICIENT FO-FC 0.901 CORRELATIONCOEFFICIENT FO-FC FREE 0.727 RMS DEVIATIONS FROM IDEAL VALUES COUNT RMSWEIGHT BOND LENGTHS REFINED 4628 0.014 0.022 ATOMS (A) BOND ANGLESREFINED ATOMS 6285 1.738 1.953 (DEGREES) TORSION ANGLES, PERIOD 1 5778.885 5.000 (DEGREES) TORSION ANGLES, PERIOD 2 195 40.409 24.205(DEGREES) TORSION ANGLES, PERIOD 3 770 23.636 15.000 (DEGREES) TORSIONANGLES, PERIOD 4 22 18.930 15.000 (DEGREES) CHIRAL-CENTER RESTRAINTS 7040.110 0.200 (A**3) GENERAL PLANES REFINED 3478 0.007 0.021 ATOMS (A)

TABLE 8 IFN-α8-hzACO-1 Fab L chain interactions. IFN-α8 Atoms hzACO-1Fab L Atoms Distance (Å) Leu 118I CB Tyr 95L CE2 3.78 Leu 118I CG Tyr95L CE2 3.68 Tyr 95L CD2 3.90 Leu 118I CD1 Tyr 95L CG 3.95 Tyr 95L CE23.53 Tyr 95L CD2 3.21 Ser 93L O 3.70 Tyr 95L N 3.96 Phe 97L CE1 3.62 Leu118I CD2 Trp 92L O 3.84 Arg 121I NH2 Tyr 95L OH 3.93* Lys 122I CA Tyr33L OH 3.38 Lys 122I CB Tyr 33L OH 3.12 Lys 122I CG Tyr 33L CE1 3.75 Tyr33L CZ 3.37 Tyr 33L OH 2.93 Lys 122I CD Tyr 33L CZ 3.74 Tyr 33L OH 3.40Lys 122I CE Tyr 33L OH 3.33 Lys 122I C Tyr 33L OH 3.70 Lys 122I O Tyr33L OH 3.41* Gln 125I CG Trp 92L CH2 3.18 Tyr 33L CE1 3.80 Trp 92L CZ23.63 Gln 125I CD Trp 92L CH2 3.70 Tyr 35L OH 3.81 Trp 92L CZ2 3.58 Gln125I OE1 Tyr 35L OH 3.50* Gln 125I NE2 Ser 32L O 3.24*** Ser 51L OG3.37* Tyr 33L CE1 3.94 Tyr 35L OH 3.59* Trp 92L CZ2 3.67 Arg 126I CD Tyr33L OH 3.94 Leu 129I CD1 Ser 51L CB 3.80 Ser 51L OG 3.77 Ser 32L OG 3.40Leu 129I CD2 Ser 51L OG 3.90 Thr 132I OG1 Tyr 50L OH 3.90* A cut-off of4.0 Å was used. The contacts were identified by the CONTACT computerprogram of the CCP4 suite. In the last column “***” indicates a strongpossibility for a hydrogen bond at this contact (distance <3.3 Å) ascalculated by CONTACT, “*” indicates a weak possibility (distance >3.3Å). Blank indicates that the program considered there to be nopossibility of a hydrogen bond.

TABLE 9 IFN-α8-hzACO-1 Fab H chain interactions. IFN-α8 Atoms hzACO-1Fab H Atoms Distance (Å) Ser 55I CB Arg 57H NH2 3.58 Ser 55I OG Arg 57HNE 3.18*** Arg 57H CZ 3.30 Arg 57H NH2 2.80*** His 58I CG His 55H CD23.73 His 58I CE1 Asn 52H ND2 3.94 Ser 54H CB 3.90 Ser 54H OG 3.79 His58I NE2 His 55H CD2 3.53 Ser 54H CB 3.60 Ser 54H OG 3.20*** His 58I CD2His 55H CD2 3.19 Glu 59I CD Trp 33H NE1 3.90 Glu 59I OE2 Trp 33H CD13.74 Trp 33H NE1 3.01*** Gln 62I CD Asn 52H OD1 3.44 Thr 30H O 3.71 Gln62I OE1 Asn 52H CG 3.91 Asn 52H OD1 3.02*** Ser 54H CB 3.67 Thr 30H C3.69 Thr 30H O 2.55*** Asn 31H CA 3.87 Gln 62I NE2 Asn 52H CG 3.77 Asn52H OD1 3.13*** Asn 52H ND2 3.95* Gln 63I CD Leu 101H CD1 3.83 Gln 63IOE1 Leu 101H CG 3.87 Leu 101H CD1 2.96 Gln 63I NE2 Leu 101H CD2 3.79 Asn66I CG Asn 31H O 3.94 Asn 66I OD1 Tyr 32H CE1 3.54 Asn 31H CB 3.89 Asn31H C 3.86 Asn 31H O 2.80*** Tyr 32H CZ 3.82 Asn 66I ND2 Asn 31H OD13.79* Glu 97I CG Ser 54H OG 3.85 Glu 97I CD Ser 54H O 3.97 Ser 54H OG3.24 Glu 97I OE1 Ser 54H O 3.54* Glu 97I OE2 Ser 54H CB 3.88 Ser 54H OG2.56*** Arg 121I CZ Glu 50H CD 3.73 Glu 50H OE1 3.31 Glu 50H OE2 3.30Arg 121I NH1 His 35H CE1 3.89 Glu 50H CD 3.78 Glu 50H OE1 3.06*** Glu50H OE2 3.66* Trp 105H CZ3 3.82 Arg 121I NH2 Glu 50H CD 3.08 Glu 50H OE12.80*** Glu 50H OE2 2.73*** Trp 33H CG 3.66 Trp 33H CE2 3.33 Trp 33H CD23.51 Trp 33H CZ2 3.88 Trp 33H CD1 3.54 Trp 33H NE1 3.35* Phe 124I CB Leu101H CD1 3.79 Gln 125I CD Gly 102H N 3.69 Gln 125I OE1 Leu 101H CA 3.76Leu 101H CB 3.38 Leu 101H C 3.57 Gly 102H N 2.55*** Gly 102H CA 3.24 Thr128I OG1 Leu 101H CB 3.84 A cut-off of 4.0 Å was used. The contacts wereidentified by the CONTACT computer program of the CCP4 suite. In thelast column “***” indicates a strong possibility for a hydrogen bond atthis contact (distance <3.3 Å) as calculated by CONTACT, “*” indicates aweak possibility (distance >3.3 Å). Blank indicates that the programconsidered there to be no possibility of a hydrogen bond.

Example 7 Design of Structure Based Mutations for Affinity Maturation ofhzACO-1

If the epitope and paratope of an antibody/antigen complex is not known,a large number of possible amino acid residues may be prone formutations in order to improve the affinity of an antibody and moreover,they can be converted into any of the 20 remaining amino acid residuesto identify the optimal residue at the particular position. The CDRregion holds approximately 54 residues available for mutations. Thus, inorder to improve the affinity of the interaction 54×20=1080 analoguesmay be generated only within the CDRs. In addition, mutations outsidethe CDRs may be made in order to improve affinity.

However, when the structure of the antibody/antigen is know, a morelimited number of qualified mutations that improve affinity may bepredicted and analysed, based on structural predictions. Accordingly,based on the crystal structure of IFN-α8 in complex with the Fabfragment of hzACO-1, as described in Example 6, three mutants thatimprove hzACO-1 binding to all IFN-α subtypes were identified. The 3mutants are hzACO-1 HC T30R, hzACO-1 LC Y32E and the combined hzACO-1Y32E, T30R (residue numbering according to Kabat). The identification ofthe mutants is described below.

LC Y32E: It can be seen that the electron density for residues Lys 122of IFN-α8, which is part of the binding epitope to hzACO-1, indicates arather high mobility. Moreover, the atom Oη of residue Tyr 32 (Kabatnotation) of the hzACO-1 light chain is directed towards the Cβ and Cγatoms of the Lys 122 side chain. That interaction is not any optimalresidueresidue interaction. Mutating the light chain Tyr 32 to anegatively charged residue like Glu, or Asp, make the possibility offorming a strong ionic bond between the antibody light chain Tyr 32residue and the positively charged Lys 122 residue of IFN-α8. For thatreason Y was exchanged for E in order to improve the affinity of thehzACO-1 antibody.

HC T30R: For each hzACO-1 residue close to IFN-α8 in the X-raystructure, the following properties were calculated: number of core sidechain atoms (core), number of periphery sidechain atoms (peri), numberof charged interactions (char), number of hydrogen bonds (hybo) andnumber of hydrophobic interactions (hyph) and given in Table 10 below:

TABLE 10 Properties of amino acids of the hzACO-1 mAb Kabat Res coreperi char hybo hyph Light Chain 29 D 1 2 1 31 S 2 1 32 Y 5 3 6 34 Y 3 249 Y 3 1 1 2 50 S 2 8 53 N 1 3 1 91 W 5 2 9 92 S 1 93 S 4 94 Y 6 2 96 F2 2 Heavy Chain 28 T 3 30 T 3 31 N 4 1 32 Y 6 2 1 33 W 10 3 35 H 2 2 50E 4 1 1 1 52 N 3 1 1  52A P 1 53 S 2 2 54 H 4 2 6 56 R 5 2 1 1 58 I 2 22 64 Q 1 71 R 1 1 97 L 4 3 98 G 1 99 P 2 100A W 2 3 1

The number of core and periphery atoms were calculated by overlaying theX-ray structure onto a 101×101×101 node grid and for each grid pointcalculating the number of atoms <2.5 Å (N_(ex)) and atoms <3.5 Å(N_(in)) from the node. Core nodes have N_(ex)>0 covering atoms fromboth hzACO-1 and IFN-8. Periphery nodes have N_(ex)=0 and N_(in)>0covering atoms from both hzACO-1 and IFN-α8. Core sidechain atoms arenow atoms <2.5 Å from any node. Periphery sidechain atoms are now atoms≧2.5 Å & <3.5 Å from any node.

Finally periphery residues are defined as residues with only peripheryatoms and no interactions, so they can be modified to create binding. LCS92, HC T28, HC T30, HC P52, HC Q64 and HC P99 have be identified.Focusing on heavy chain non-prolines, it is seen by visual inspectionthat the mutation HC T28R would have a possible interaction with D90, HCT30R would have a possible interaction with both D90 and E97 and Q64Rwould have a possible interaction with E114, but other similar mutationscan also be applied. Since only E97 is conserved across all interferonalphas, HC T30R is expected to give the best binding with a similarprofile as hzACO-1.

The specific mutation HC T30R was designed to establish a charge-chargeinteraction in the periphery of the binding site to improve the affinityof hzACO-1.

Furthermore, a double mutant containing both the LC Y32E and the HC T30Rmutations, termed hzACO-1 Y32E, T30R was generated and analyzed todetermine if the two mutations would have additive effects.

Example 8 Determination of the Kinetic Parameters for the InteractionBetween hzACO-1, hzACO-1 Variants and Recombinant Human IFN-α Subtypes

Protein interactions can be monitored in real-time using surface plasmonresonance (SPR) analysis. In this study, SPR analysis was performed onBiacore® 3000 and Biacore® T100 instruments in order to characterize theanti-IFNα monoclonal antibody hzACO-1 and variants thereof, with respectto affinity towards various subtypes of recombinant human Interferonalpha (IFN-α).

Affinity studies were performed using a direct binding procedure, withthe respective monoclonal antibody covalently coupled via free aminegroups to the carboxymethylated dextrane membrane (CM5) on the sensorchip surface. Recombinant IFN-α subtypes (PBL Biomedical Laboratories,NJ, USA.) were injected in various concentrations, followed by adissociation period with constant buffer flow over the sensor chipsurface. Using this experimental design, the binding of IFN-α to theimmobilized monoclonal antibody can be regarded as a 1:1 binding, withone IFN-α molecule binding to one antibody binding site. The kineticparameters for the interaction can be calculated using a 1:1 interactionLangmuir fitting model.

The purified monoclonal antibodies were immobilized in individual flowcells on a CM5 type sensor chip. Immobilizations were performed using astandard amine coupling procedure, aiming for an immobilization level of1000 Resonance Units (RU).

HBS-EP pH 7.4 (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.005%Polysorbate P20) was used as running buffer, and diluent for therecombinant IFN-α's. Association (injection) was 4 min., followed by a12 to 30 min. dissociation period. Flow rate was 50 μl/min. Experimentswere performed at 25° C. Detection in all flow cells simultaneously.Flow cell #1 contained no immobilized antibody, and was used forsubtraction of background and bulk.

The kinetic parameters were calculated by global fitting of the data fora given antibody—antigen combination using a 1:1 Langmuir binding model.Data was inspected for mass-transport limitations prior to calculationof the kinetic parameters.

Experiments were performed on Biacore® 3000 and T100 instruments. Datawas evaluated using Biaeval™ 4.1 and Biacore® T100 evaluation software.

The kinetic parameters obtained are valid only in the buffer used, andwith the recombinant form of the antigen.

Results

In order to generate a humanized ACO-1 antibody with retained affinity anumber of humanized variants were generated as described in example 2, 3and 7, using different strategies. The kinetic parameters for theinteraction between recombinant human IFN-α subtypes and variants ofhzACO-1 was obtained by SPR analysis. As seen in Table 11 even thoughhzACO-1, generated as described in Example 2 contains a truncated CDR H2and no backmutations, the affinity of the hzACO-1 has been retained ascompared to the murine ACO-1, as shown by the KD of the hzACO-1 beingwithin two-fold of the mouse antibody. Accordingly, no furtherbackmutations from human to mouse ACO-1 in the framework regions wererequired for humanization, as identified and described in example 2. Inaddition, the IFN-α subtype profile of the hzACO-1 antibody had beenretained, as shown in Table 2.

TABLE 11 Kinetic parameters for the interaction of recombinant IFN-αAwith ACO-1 and hzACO-1 variants. KD mAb/KD Anti-IFN-α mAb KD (M) ka(1/Ms) kd (1/s) hzACO-1 ACO-1 3.09E−09 1.24E+05 3.75E−04 0.60 hzACO-14.46E−09 1.24E+05 5.56E−04 1 hzACO-1-L33F 4.86E−09 1.89E+05 9.18E−041.17 hzACO-1-S50G 4.94E−09 1.98E+05 9.78E−04 1.19 hzACO-1-T28S 2.97E−091.58E+05 4.68E−04 0.63 hzACO-1-N31S 2.20E−09 1.16E+05 2.55E−04 0.61hzACO-1-I58S 1.93E−08 1.93E+05 3.74E−03 4.09 hzACO-1-S76N 5.07E−091.16E+05 5.88E−04 1.09 hzACO-1-T77I 6.79E−09 9.80E+04 6.65E−04 1.46hzACO-1-A93V 2.16E−09 1.08E+05 2.34E−04 0.60 hzACO-1-T28S, 2.22E−091.56E+05 3.45E−04 0.77 N31S hzACO-1-N31S, 1.51E−09 1.59E+05 2.40E−040.52 A93V hzACO-1-T28S, 1.66E−09 1.61E+05 2.68E−04 0.69 A93VhzACO-1-T28S, 1.57E−09 1.85E+05 2.91E−04 0.65 N31S, A93V (KD is theequilibrium dissociation constant, ka is the association rate constantand kd is the dissociation rate constant.)

The kinetic parameters for the interaction of hzACO-1 as compared to ahumanized ACO-1 molecule generated by traditional CDR grafting andaccordingly contains a large murine CDR H2 (designated hzACO-1-kabatCDRH2), with recombinant human IFN-αA are listed in Table 12. As shown,the affinity of the hzACO-1 molecule, humanized as described in Example2 and containing a shorter CDR H2 than the hzACO-1-kabat CDRH2 molecule,were equal, showing that the humanization process described in Example 2generates a humanized variant as good as that generated by simple CDRgrafting, while having a more human sequence.

TABLE 12 Kinetic parameters for the interaction of recombinant IFN-αAwith hzACO-1 and hzACO-1-kabat CDRH2. KD mAb/ mAb KD (M) ka (1/Ms) kd(1/s) KD hzACO-1 hzACO-1 1.9E+09 2.22E+05 4.15E−04 1 hzACO-1-kabat1.7E+09 2.05E+05 3.41E−04 0.9 CDRH2 (KD is the equilibrium dissociationconstant, ka is the association rate constant and kd is the dissociationrate constant.)

Furthermore, in order to investigate if the hzACO-1 and thehzACO-1-kabat CDRH2 had comparable kinetic parameters of the binding tovarious subtypes of human IFN-α, a dissociation comparison experimentwas performed on selected human IFN-α subtypes (Table 13). This showsthat the affinity of the hzACO-1 appears to be retained as compared tohzACO-1-kabat CDRH2 to all tested subtypes despite having a shortermouse CDR H2 sequence.

TABLE 13 Comparison of dissociation rate constants (kd) of theinteraction between various subtypes of recombinant human IFN-αA withhzACO-1 and hzACO-1-kabat CDRH2 respectively. mAb hzACO-1-kabathzACO-1/hzACO- hzACO-1 CDRH2 1-kabat CDRH2 Subtype kd (1/s) kd (1/s)Ratio IFN-αH2 4.13E−04 3.28E−04 1.26 IFN-αK 2.89E−04 3.41E−04 0.85IFN-α4b 2.17E−04 1.55E−04 1.40 IFN-αWA 2.91E−04 3.66E−04 0.80

In order to try to improve the affinity of hzACO-1 beyond that of themouse ACO-1 antibody, the kinetic of the parameters between IFN-αA anddifferent hzACO-1 variants containing mutations based on the ACO-2sequence were measured (Table 11). In order to correlate the parametersobtained in the separate experiments performed, the KD value of eachindividual antibody was normalized against that of hzACO-1 in the sameexperiment, and the relation of the KD value of the individual mAb tothe KD of hzACO-1 in the same experiment is shown in the column “KD mAbvs. KD hzACO-1”.

The affinity determination further demonstrated KD values of all ACO2derived hzACO-1 antibody variants (except hzACO-1-I58S) in the lower nMrange. The minor variations in the KD values are predominantly relatedto differences in the kd. Introduction of the single amino acidsubstitutions N31S, A93V, or T28S, and combinations thereof, in thehzACO-1 slightly increased the affinity to a level similar to that ofACO-1. The hzACO-1-I58S mutation has a pronounced negative effect on thekd value, demonstrating the importance of this particular amino acid forthe stability of the hzACO-1/IFNα-A complex.

Based on the hzACO-1 Fab/IFN-α8 crystal structure, a number of aminoacid substitutions were introduced in hzACO-1 in order to increase theaffinity even further (see Example 7). Of these, two singlesubstitutions, Y32E of the light chain and T30R of the heavy chain, hadsignificant positive effects on the affinity (Table 14) increasing theaffinity approximately 2 and 6 fold respectively, against IFN-αA.Remarkably, by combining the two mutations the hzACO-1 constructcontaining both the Y32E and T30R an approximately 10 fold increase inaffinity was observed against IFN-αA (Table 15).

TABLE 14 Kinetic parameters for the interaction of recombinant IFN-αAwith hzACO-1 and hzACO-1 variants respectively. KD mAb/ mAb KD (M) ka(1/Ms) kd (1/s) KD hzACO-1 hzACO-1 3.20E−09 1.39E+05 4.43E−04 1 hzACO-1LC Y32E 1.54E−09 2.47E+05 3.81E−04 0.5 hzACO-1 HC T30R 5.40E−10 1.31E+057.08E−05 0.16 (KD is the equilibrium dissociation constant, ka is theassociation rate constant and kd is the dissociation rate constant.)

TABLE 15 Kinetic parameters for the interaction of recombinant IFN-αAwith hzACO-1 and hzACO-1 Y32E, T30R respectively. KD mAb/ mAb KD (M) ka(1/Ms) kd (1/s) KD hzACO-1 hzACO-1 2.72E−09 1.78E+05 4.85E−04 1 hzACO-1Y32E, 2.97E−10 1.49E+05 4.43E−05 0.1 T30R (KD is the equilibriumdissociation constant, ka is the association rate constant and kd is thedissociation rate constant.)

The kinetic data in Table 15 illustrate, that a hzACO-1 Y32E, T30Rconstruct generated based on a rational design approach, had retainedits IFN-α subtype profile as it does not bind to IFN-α1 but does bind tothe remaining subtypes tested. In order to validate that the effect onthe kinetic parameters caused by the Y32E, T30R mutations were reflectedin the binding to various subtypes of human IFN-α, a dissociationcomparison experiment was performed on selected human IFN-α subtypes(Table 16). Although the suggested mutations of the double mutanthzACO-1 construct were based on the structure of the IFN-α8,unpredictably the improved off-rates were observed for all tested humanIFN-α subtypes varying between 6-64 fold.

TABLE 16 Comparison of dissociation rate constants (kd) of theinteraction between various subtypes of recombinant human IFN-α withhzACO-1 and hzACO-1 Y32E, T30R respectively. mAb hzACO-1/hzACO-1 hzACO-1hzACO-1 Y32E, T30R Y32E, T30R Subtype kd (1/s) kd (1/s) Ratio IFN-αA6.24E−04 6.85E−05 9.1 IFN-α1 No binding No binding — IFN-α4b 1.12E−031.74E−05 64.4 IFN-αI 1.55E−03 3.15E−05 49.2 IFN-αJ1 4.54E−03 1.95E−0423.3 IFN-αWA 2.47E−03 4.05E−04 6.1

Example 9 Analysis of hzACO-1 Constructs in a CPE-assay

This example shows that hzACO-1 was able to inhibit the protectiveeffect of all IFN-α subtypes tested, except for those of IFN-α1 andIFN-αD, which were unaffected by hzACO-1.

Materials & Methods

This anti-IFN-α neutralization assay used is based upon the lytic effectof EMC virus on A549 cells. All IFN-α subtypes can inhibit the EMC virusreplication in A549 cells, resulting in cell survival, which can bemeasured as cellular DNA staining. The neutralizing effect of ananti-IFN-α antibody on different IFN-α subtypes can be measured bydiminished cellular DNA staining, corresponding to increased cell lysis.

The assay was performed in plates with 96 wells (Nunc® Co., Cat. No.167008), where each well contained a final volume of 200 μL. All IFN-αpreparations were from PBL Biomedical Laboratories, NJ, USA.

To each well, four solutions were added (IFN-α, hzACO-1, cells andvirus) at a volume of 50 μL each. All solutions were prepared in F12Kaighn's medium (Gibco® Cell Culture, Cat. no. 21127) with 10% FCS. Thespecific concentration of each IFN-α subtype (listed in Table 17 below)was derived from previous studies. The antibody concentrations used inthe assay were selected based on existing data obtained from use of,e.g., murine antibodies ACO-1.5.2 and ACO-2.2.

Each IFN-α subtype was pre-incubated with hzACO-1 for 2 hours at 37° C.,5% CO₂. The anti-interferon antibody was diluted as shown in Table 17below. After pre-incubation of antibody with IFN-α, 50 μL ofcell-solution (300000 cells/mL) were added to obtain 15000 cells/well.After 4.5 hours incubation at 37° C., 5% CO₂, 50 μL EMC virus at aconcentration of 10^3.5 TCID₅₀ were added, followed by incubation for 48hours at 37° C., 5% CO₂.

The supernatant was subsequently carefully removed, and 50 μL crystalviolet solution 0.5% crystal violet, 25% methanol) were added. After 15min of incubation at room temperature, the wells were washed in waterand dried overnight.

To the dried plates were then added 200 μL/well of pure methanol for 15min to extract the crystal violet from the cells. After extraction, 100μL of the supernatant were carefully transferred to a new 96-well plate(Nunc® Co., Cat. No. 256510), and 100 μL Milli-Q® water added to eachwell. The plate was then measured in an ELISA reader at 590 nm.

The raw data retrieved from the ELISA reader was corrected for methanoland plate background prior to analysis.

TABLE 17 CPE assay parameters. IFNα Subtype [IFNα] (pg/μL)* [hzACO-1](ng/mL) IFN-αA 1.25 × 10−1  2.500->0 IFN-α2 3.125 × 10−2  2.500->0IFN-αF 6.25 × 10−2  2.500->0 IFN-αK 6.25 × 10−2  2.500->0 IFN-αWA 6.25 ×10−2  2.500->0 IFN-αB2 2.5 × 10−2 2.500->0 IFN-αH2 1.25 × 10−1  2.500->0IFN-αI 5.0 × 10−2 2.500->0 IFN-αJ1 1.0 × 10−1 2.500->0 IFN-α4a 2.5 ×10−1   250->0 IFN-αC 2.5 × 10−2   250->0 IFN-αG 2.5 × 10−1   250->0IFN-α4b 1.25 × 10−1    250->0 IFN-αD 3.75 50.000->0  IFN-α1 1.0 50.000->0 Results and Discussion

Six different controls were used on all plates in the study (cells,cells+antibody, cells+IFN-α, cells+antibody+IFN-α, cells+virus, andcells+IFN-α+virus) to ensure that the cytopathic effect observed in theassay was not caused by cytotoxicity of the antibody and/or IFN-α, northat any lack of IFN-α-protection of the cells against the virus wascausing the cytopathic effect. No significant cytotoxic effect wasobserved in the assays, and at no level of interferon was there a signof a cytopathic effect in the controls.

As shown in FIG. 4, this CPE-assay showed that the protective effect ofalmost all interferon subtypes could be inhibited by hzACO-1. However,the protective effects of IFN-αD and IFN-α1 were not inhibited byhzACO-1, even at antibody concentrations of 50000 ng/mL. Accordingly,the specificity of the mouse ACO-1 was retained in the hzACO-1construct.

Example 10 Analysis of ACO-Derived Antibodies in a Reporter Gene (RG)Bioassay

A luciferase-based reporter gene assay was utilized to evaluate theability of hzACO-1 antibody variants to neutralize the biologicalactivity of recombinant IFNα subtypes.

Materials

Dulbecco's Modified Eagle's Medium “complete”: DMEM incl. phenol red+10%FCS+2 mM L-glutamine+penicillin+streptomycin+2-me., Nunc® Co. 96-welloptical bottom plate, black tissue culture treated, PBS including 1 mMCa²⁺ and 1 mM Mg²⁺, Steady-Glo® Luciferase assay system (Promega®Corp.).

The 93D7 cell line was derived by stable transfection of the A549 cellline (CLL-185, ATCC) with an IFN-inducible construct, harboring the Mxpromoter driving a luciferase reporter gene. The Mx promoter consists ofa 1.6 kb BamHI fragment containing the murine MxA promoter and IFNresponse elements excised from pSP64-Mxp (PstI-PvuiI)-rβglo (Lleonart etal. (1990) Biotechnology 8: 1263-1267).

The IFN-α subtypes (all from PBL Biomedical Laboratories, NJ, USA) usedare listed in FIG. 5.

Methods

RG assays were performed in quadruplicate wells in opaque Nunc™ Co.96-well optical bottom plates. For every assay, a positive control IFNαwas included as well as negative control wells containing non-stimulatedcells and cells treated with antibody in the absence of IFNαstimulation.

Specifically, adherent 93D7 cells were harvested from flasks by removingthe culture media, washing once with PBS, and trypsinizing.Trypsinization was stopped using DMEM complete. Cells were counted andadjusted to 600,000/ml in complete DMEM.

Purified anti-IFN-α mAbs were pre-incubated with recombinant IFNsubtypes for 1 h in a total volume of 100 μl DMEM complete at 37° C.+5%CO₂. Following antibody-IFN incubation, 50 μl 93D7 cells were added andincubated for 5 h at 37° C.+5% CO₂.

To assay for MxA-driven luciferase induction by recombinant IFNsubspecies, the concentration of IFN was adjusted to contain the amountto be placed in each well in 100 μl. 100 μl of IFN was placed inquadruplicate wells, incubated for 1 h at 37° C.+5% CO₂. Subsequently,50 μl cells were added and incubation continued for additionally 5 h.

To assay for inhibition of MxA-driven luciferase induction byrecombinant IFN subspecies using purified mAbs, 50 μl of a desireddilution of recombinant IFN was added per well. 50 μl of antibodydiluted in DMEM complete was then added to the wells and incubated for 1h at 37° C.+5% CO₂. After this incubation, cells were added andincubation continued for additionally 5 h.

After the 5 h incubations, medium was carefully removed from the cellsusing a multichannel pipet. Next, an adhesive black blocker was affixedto the bottom of the 96 well plate. 100 μl PBS with Ca²⁺ and Mg²⁺ ionswas added to each well. 100 μl reconstituted SteadyGlo® reagent wasadded to each well, making sure that contents in each well were mixedthoroughly. The plates were sealed with a clear adhesive strip.Following 5 min. incubation at room temperature in the dark,luminescence was read in a Topcount® luminescence counter (Perkin Elmer®Inc.).

For calculation of the degree of inhibition exerted by the antibody toIFN induced (MxA driven) luciferase activity, counts were when comparingAb inhibition of various IFN-α subtypes normalized to activity levels inthe absence of antibody and this value was set to 100%. For comparingvariant form of antibody inhibition of single IFN-αs data are shown asraw luciferase counts. IFNs were initially titrated in the absence ofantibody to determine the EC50 and the IFN concentration at whichplateau in the assay was reached. When testing for antibody inhibition,IFN was generally used at 80% of maximum stimulation levels to ensureboth a solid induction of luciferase as well as operating at a levelbelow saturation in the assay. Prism® software (Graph Pad Software,Inc., San Diego) was used for calculations and data display.

Results and Discussion

Humanized ACO-1 constructs were evaluated for their ability to inhibitvarious human IFN-α subtypes in the reporter gene assay. FIG. 5 showsnormalized data for the inhibition of 12 IFN-α subtypes by the hzACO-1antibody. IFN-α stimulation in the absence of antibody was set to 100%whereas mock treated cells (receiving medium only) was set to 0%. Datapoints are shown with standard error. Curves were calculated as best fitsigmoidal response curves using the Prism® software. hzACO-1 was capableof inhibiting all tested subspecies of IFN-αs except for IFN-αD, andaccordingly the specificity of the parent mouse ACO-1 antibody wasretained during humanization in the hzACO-1 antibody. Inhibition wascomplete in that, at high antibody concentrations, IFN-α activity wasreduced to background levels. IC50 for the inhibition of the variousIFN-α subtypes ranged in this study from 28 ng/ml to 314 ng/ml. IFN-αDcould not be inhibited even at higher hzACO-1 concentrations.

FIG. 10 shows a comparison of the mouse ACO-1 Ab to the humanized ACO-1(hzACO-1) as well as two variants hereof in the RG assay. One variant isa humanized ACO-1 harboring a the entire CDRH2 (designated hzACO-1-kabatCDRH2) whereas the hzACO-1 was constructed with a shorter CDRH2 asdescribed in example 2. In addition the figure shows another mutatedhzACO-1 which has been optimized for interaction with IFN-αs (designatedhzACO-1 Y32E, T30R) through rational design, as described in Example 7.These four recombinant mAb variants were compared with respect toinhibition of five different representative IFN-α subtypes in the RGassay.

hzACO-1 displayed for all five IFN subtypes almost comparable IC50values as mouse ACO-1, quantitatively the IC50s being less than twofold(see Table 18 for relative IC50 values) in accordance with the observedKDs reported in Example 8. The humanization was thus accomplished withan affinity loss less than twofold of the parent ACO-1 antibody forfunctional inhibition. In conclusion, the mAb humanization of ACO-1 hasproduced an antibody that has retained the affinity for IFN-αs.Accordingly, both the affinity and the potency was considered retainedin of the hzACO-1 antibody as compared to the original mouse ACO-1, andno further backmutations were required.

As described in Example 2, the humanization method of ACO-1 resulted inan antibody with relatively more human amino acids in the CDR H2 ascompared to commonly humanization by simple CDR grafting. Whereas thiscould be expected to lead to reduced potency in the humanized ACO-1 forneutralization of IFN-α subtypes, as shown in FIG. 10 and Table 18,suprisingly this is not the case, as the hzACO-1 and hzACO-1-kabat CDRH2are equipotent for all the IFN-α subtypes tested. This is in agreementwith the affinities reported for the two ACO-1 variants as described inExample 8.

TABLE 18 IC50 values for IFN-α subtypes inhibition by hzACO-1 variants.IC50 values relative to hzACO-1 IFN- mAb Variant IFN-αA IFN-αB2 IFN-αFIFN-αG αJ1 hzACO-1 1.00 1.00 1.00 1.00 1.00 ACO-1 0.60 0.47 0.82 0.560.58 hzACO-1 Y32E, T30R 0.03 0.02 0.05 0.06 0.02 hzACO-1-kabat CDRH20.95 0.88 0.95 0.78 1.15 Normalized data. IC50 values in were for eachIFN-α species normalized to that of hzACO-1. A value less than onetherefore indicates a more potent antibody than hzACO-1 inhibition ofIFN-α activity, whereas conversely values higher than one indicatesinhibition with lesser potency.

To determine the potency of ACO-2 derived hzACO-1 variants, as describedin Example 3, these were compared to hzACO-1 using the RG assay.Comparisons were made using two IFN-α subspecies (IFN-αF and IFN-αA).Four different single amino acid substitutions were tested: T28S (i.e.,at position 28, (according to Kabat) threonine was substituted withserine), I58S, N31S, and A93V.

Whereas the I58S variant inhibited the IFN-action with reduced potency,and possibly also with reduced efficacy (IFN-αA), the T28S and N31Svariants both displayed potencies similar to that of hzACO-1. The A93Vsubstituted variant, however, showed increased potency for inhibition ofIFN-effects as measured in the RG assay (FIG. 6). Although differencesin the effects of the substitutions could be detected between differentIFN-α subtypes, the trend was the same for the two forms in all fourcases.

Through rational design using the crystal structure in Example 6, amutant was constructed having two amino acids changed, HC T30R, LC Y32Eof hzACO-1 (designated ACO-1 Y32E, T30R), as described in example 7, toimprove binding to IFN-α. Even though the mutations were based on thestructure of IFN-α8, as seen from FIG. 10 (A-E) surprisingly this mutanthad increased potency for inhibition of all the tested IFN-αs.Furthermore, Table 17 shows that whereas the increment in potency isdependent on the specific subtype it is in the order of 16 fold and uptil 50 fold improvement of potency.

It follows that the epitope information obtained from the crystalstructure in Example 6 can be used to design other antibody variantswith improved binding affinity to this epitope. It also follows thatsuch humanized antibody variants according to the present invention areembranced by the scope of the present invention.

Example 11 Protein Characterization of Humanized ACO-derived Antibodies

This Example concerns the thermal stability of hzACO-1 and differentvariants.

Materials

-   -   Antibodies    -   hzACO-1 expressed as with human IgG1, IgG2 and IgG4 isotypes    -   hzACO-1-T28S    -   hzACO-1-N31S    -   hzACO-1-A93V    -   hzACO-1-T28S-N31S

The following is a list of the buffers (100 mM) and their pH values usedin the study: citric acid/sodium citrate, pH 3.0, 3.5; sodium acetate,pH 4.0, 4.5, and 5.0; histidine, pH 6.0, 6.5; imidazole, pH 7.0;glycine-glycine pH 8.0, 9.0, 10.0.

The following is a list of additives and their concentrations used inthe study: NaCl, 100 mM; sucrose 0.25 M, 0.50 M; phenol, 0.5%; Tween 80,0.01%; glycerol 10%.

Thermofluor Stability Measurements

Solutions of 10 μl 400× SYPRO® Orange protein gel stain 5000×Concentrate in DMSO (Invitrogen® Corp. Molecular Probes), 25 μl bufferand 10 μl protein (10 μM) were added to wells of a 96-well PCR-plate(Bio-Rad® Laboratories, Inc.). The plates were sealed with Microseal BAdhesive sealer MSB-1001 (Bio-Rad® Laboratories, Inc.) and heated in aMyiQ Single-Color Real-Time PCR Detection system (Bio-Rad® Laboratories,Inc.) from 25 to 95° C. in increments of 0.5° C. Fluorescence changes inthe wells of the plate were monitored simultaneously with acharge-coupled device (CCD) camera. The wavelengths for excitation andemission were 490 and 575 nm, respectively. The midpoint temperature forprotein unfolding transition was determined as the first derivativemaximum of the fluorescence intensity as a function of temperature

The thermofluor graphs for hzACO-1 and variants showed the two expectedtemperature transitions for IgG proteins, reflecting the two maindomains, Fc and Fab. The proteins showed a lower Tm at lower pH. AbovepH 5.5 the transition midpoint was rather constant.

The effect of additives was the same for the different antibodies.Generally, sucrose at 0.5M had the most stabilizing effect, whileaddition of NaCl, phenol and Tween® 80 detergent seemed to have adestabilizing effect.

At lower pH (pH 3.5), a difference in stability between hzACO-1 and thedifferent variants was observed (FIG. 7A). The double mutanthzACO-1-T285-N31S and single mutant hzACO-1-A93V showed the higheststability at this pH. This could be important in a purification processfor a therapeutic antibody product, as virus inactivation steps oftenare carried out at pH 3.5-4.0. At higher pH (pH 4.5, 5.5; FIGS. 7B and7C, respectively) this difference in stability decreased.

Solution Stability Study

Solutions of hzACO-1-IgG4, hzACO-1-IgG1 and hzACO-1-IgG2 in 15 mMhistidine pH 6.5, sucrose 20 mg/ml and Tween® 80 detergent at 0.01% wasincubated at 40° C. and samples were withdrawn for analysis at initialand after 5 weeks. Distribution of intact protein, soluble aggregateand/or fragments of hzACO-1 was determined using size exclusionchromatography (SEC-HPLC). A high-performance liquid chromatography(HPLC) system model 1100 or 1200 liquid chromatography system (AgilentTechnologies®, Palo Alto, Calif.) was used with a BIOSEP® SEC 3000(Phenomenex®) column at a flow rate of 0.8 mL/min using pH 7.2 and withphosphate-buffered saline (PBS) as the mobile phase. Protein wasdetected by monitoring the OD at 215 nm. The percentage of each peak intotal protein area was calculated.

The amount of formed high molecular weight forms of the hzACO-1-isotypesis shown in FIG. 11 and shows clearly that the IgG4 construct showed noaggregate formation during the incubation while both the IgG1 and IgG2isotypes formed high molecular weight variants.

Furthermore, the hzACO-1-IgG1 variant showed a low molecular weightfragment after incubation. The amount of the fragment was 1.3%.

Accordingly, from a stability point of view the hzACO-1 IgG4 is a moreattractive therapeutic molecule than the corresponding IgG2 and IgG1antibodies.

Examples 12 ADCC Analysis

As described in example 6 the ACO-1 mAb and humanized versions hereofmay block the activity of IFN-α by inhibiting binding of IFN-α to thetype I interferon alpha receptor subunit 1 (IFNAR1). Accordingly, ahumanized therapeutic ACO-1 mAb may bind to the cell surface making acomplex consisting of the antibody, IFN-α and IFNAR2. This raises therisk of ACO-1 inducing antibody dependent cellular cytotoxicity (ADCC).

To this end a ADCC experiment was performed to asses the ability of thehzACO-1 antibody expressed as an IgG4 subtype in the presence ofIFN-α2A.

Materials and Methods.

Raji cells (human B cell line (ATCC #CCL-86)) were used as target cells.Raji cells were cultured in RPMI1640 supplemented with 10% fetal calfserum, 10 mM HEPES, 1 mM sodium pyruvate, 1 mM glutamine, 2.5 g/lglucose and 1% penicillin/streptomycin. Highly purified interferon-α2Awas used for the assay. The protein was tested in a reporter gene assayfor biological activity prior to use. Rituxan® (Nomeco A/S, Denmark) wasused as a positive control for ADCC, when using Raji cells, which is a Bcell line that expresses the B cell surface antigen CD-20.

Target cells were harvested and counted in a hemocytometer. 1.5*10⁶cells were transferred to a 15 mL tube and centrifuged. The supernatantwas completely removed and the cell pellet was resuspended in 100 μCi⁵¹Cr (Chromium-51) per 10⁶ cells (volume were adjusted according todecay table). During a 1 hour incubation period at 37° C. with IFN-α thevial was tapped every 15 minutes. Subsequently the cells were washedtwice in medium (RPMI1640, 10% FCS), and resuspended in 2 mL medium ofassay medium. 5000 ⁵¹Cr labelled cells were plated in a volume of 50 μLin 96 well plates (flat bottom). All samples were analyzed in triplicateMaximum—and minimum releases were determined in wells without effectorcells. Maximum release: 5000 target cells/well+1% Triton® X-100detergent. Minimum release: 5000 target cells/well. The effector cellswere purified from ‘buffy coats’ by Ficoll density centrifugation usingstandard techniques. Graded numbers of freshly isolated human PBMCs wereadded to each well. The following effector to target (E:T) cell ratioswere used: 10, 20, 40 and 80. In all experiments hzACO-1 was tested atsaturating concentration of 10 μg/mL (66 nM). IFN-α 2A was tested at0.5, 2.5, 5, and 10 nM. The final assay volume was 200 μL. After 4 hoursof incubation at 37° C. 30 μL of the supernatant was transferred to aLumaPlate™ microplate and let to air dry over night. The radioactivitywas determined in TopCount NXT™ microplate scintillation andluminescence counter (PerkinElmer®, Inc. USA). Data were entered intothe GraphPrism® program and the average counts per minute of triplicatesand the corresponding standard deviation were calculated.

Results.

As seen in FIG. 12, no induction of ADCC above background (cells orcells with IFN-α) could be observed for the hzACO-1 molecule, expressedas an IgG4 in the presence or absence of IFN-α. In contrast, Rituxumab,which was used as a positive control, did induce cell lysis at differentratios of effector and target cell ratios (E:T)

Example 13 Complement Binding ELISA Assay

As described in example 6 the ACO-1 mAb and humanized versions hereofmay block the activity of IFN-α by inhibiting binding of IFN-α to thetype I interferon alpha recepfor subunit 1 (IFNAR1). Accordingly, ahumanized therapeutic ACO-1 mAb may bind to the cell surface making acomplex consisting the antibody, IFN-α and IFNAR2. This raises the riskfor activating the complement system and inducing complement dependentcytotoxicity (CDC).

The purpose of the present complement binding study was to test whetherthe classical complement pathway is activated when hzACO-1 expressed asa human IgG4 isotype binds to and forms a complex with a correspondingepitope on hIFN-α. This is accomplished by using an ELISA, whichmeasures the binding of antibodies to C4. Binding of C4 indicates thatthe C1s is changed and the complement cascade has started. Once the C4has bound, the other complement components from the plasma will in turnbe activated, bind, and enzymatically cleave the next components of thecascade.

Materials and Methods.

Streptavidin-coated microtiter plates (236001, Nunc® Co.) were used asELISA plates. Biotinylated hIFN-α2A was used as antigen source and theplates were coated in 100 μl/well by 0.25 μg/ml protein diluted inwashing buffer (10 mM Na₃PO₄+145 mM NaCl+0.05% Tween® 20 detergent).This hIFN-α concentration was shown to be the optimal coatingconcentration in an ELISA. The plates were incubated for 60 min. at RTand gentle shaking and then washed five times in washing buffer, leavingthe buffer from the last wash in the plates for 30 min. to blockpossible residual binding sites on the plates. The buffer was discardedfrom the plates and 100 μl hzACO-1 mAb diluted in washing buffer wereadded to the plates at 1 μg/ml. The plates were incubated for 60 min. atRT and gentle shaking. The plates were washed five times in washingbuffer. A polyclonal anti-IgG4 pAb was used as a positive control bycrosslinking of the hzACO-1 IgG4 mAb. The anti-IgG4 pAb mAb was dilutedin washing buffer and added to the plates in serial dilutions from 32μg/ml to 32 ng/ml in 100 μl/well. Two different purifications of theanti-IgG4 pAb were used, one affinity purified antibody and one proteinA purified antibody. The plates were incubated for 60 min. at RT andgentle shaking. The plates were washed five times in washing buffer.Human plasma diluted 1:200 in plasma buffer (PBS w/0.3 mM Ca2+, 1 mMMg2+) was added at 100 μl/well. The plates were incubated for 60 min. at37° C. with gentle shaking. The plates were washed five times and mouseanti-human C4 (HYB162-02+HYB162-04, SSI), each diluted 1:2000 in washingbuffer, was added to the plates at 100 μl/well. The plates wereincubated for 60 min at RT with gentle shaking. The plates were washedfive times in washing buffer before addition of 100 μl/well ofHRP-rabbit anti-mouse IgG (Dako® product P0260 (Dako® A/S, Glostrup,Denmark)), diluted 1:1000 in washing buffer.

The plates were washed five times and all wells added 100 μl of TMBsubstrate. After about 6 min. of incubation, 100 μl of 4M H₃PO₄ wasadded to all wells to stop the enzyme reaction. The colour was measuredspectrophotometrically by a Victor™ plate reader (Wallac™)

Results.

hzAC0-1 expressed as a human IgG4 isotype, was tested for the ability tobind complement components by ELISA using plates coated with IFN-α. Thiswas visualised by detecting binding to C4 which is one of the componentsin the classical complement cascade. As shown in FIG. 13, hzACO-1 IgG4was unable to fix complement. As a positive control a polyclonalanti-IgG4 pAb was used to induce binding by cross-linking of the hzACO-1IgG4 antibody. If the hzACO-1 was cross bound with an anti-IgG4 pAb, aclear dose dependent binding of C4 to the anti IgG4 was detected.

Example 14 Selection of Antibody Isotype

When expressing a humanized monoclonal antibody, a human antibodyisotype needs to be selected for expression of the full length humanantibody. For the development of a neutralizing anti-IFN-α antibody, noFc-mediated effector functions are required.

Furthermore, although the ACO-1 derived antibodies are capable ofneutralizing IFN-α activity (Example 9 and Example 10), the bindingepitope of the ACO-1 antibody variants, as described in Example 6, iscompatible with simultaneous binding of the IFNAR2 receptor subunit andIFN-α. Accordingly, although the therapeutic hzACO-1 mAb binds to thesoluble IFN-α cytokine, the mAb may in fact be able to bind to cellsurfaces through the IFN-α/IFNAR2 complex hand cause cytotoxicity byrecruitment of Fc mediated effector functions. In addition to this, forantibodies against Type I IFNs (IFN-α and IFN-6) the selection of theantibody isotype is of particular importance, as it has been describedthat the Fc part of anti-IFN mAbs may in fact cause undesired biologicaleffects resulting in potentiation instead of neutralization of IFN incertain cell types as described below (Moll H P. et al J Immunol 180,1594-604, 2008).

In the presence of Type I IFNs, antibodies against human IFN-α or IFN-6that normally neutralize IFN activity in other cell types, insteadinduce IFN activity in human endothelial cells and PMBCs in the presenceof IFN-α or IFN-6. The antibodies used in these studies are of the mouseIgG1 isotype, which are known to cross bind to human Fc receptors. Theinduction of IFN activity is only seen for intact antibodies and not Fabor Fab2 fragments of the same antibodies and appears to be inhibited byblockade of Fc receptors with an antibody isotype control mAb.Accordingly, although the molecular mechanism of this phenomenon is notknown, it appears to require that these mAbs bind to cell surfacereceptors through the Fc part. Furthermore, this phenomenon appears tobe specific to Type I IFNs as antibodies against Type II IFN, IFN-γ, anda similar phenomenon is not observed for antibodies against the Type IIFN receptor, IFNAR (Moll H P. et al J Immunol 180, 1594-604, 2008).

In summary, for the development of a therapeutic anti-IFN mAb forneutralization of IFN-α Fc mediated effector function are not requiredand may in fact cause undesired biological effects by causingcytotoxicity or potentiating IFN activity in certain cell types inpatients treated with an anti-IFN-α mAb. Consequently, generation of ahumanized mAb that is unable to induce effector functions may becrucial.

Four different human antibody isotypes exists i.e. IgG1, IgG2 IgG3 andIgG4. Of these IgG1 and IgG3 are the most efficient in binding to Fcreceptors and complement and consequently cause activation of Fcmediated effector functions such as -dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC) (Salfeld J G.Biotechnol 25, 1369-1372, 2007). Mutations that abrogate binding ofvarious Fc receptors and C1q have been described in the literature(Hezareh M. et al J Virol 75, 12161-12168, 2001. Idusogie E E. et al. JImmunol 164, 4178-4184, 2000. Lund J. et al J. Immunol.; 147:2657-6,1991. & Chappel et al 1991, Canfield M S et al J Exp Med. 173:1483-91,1991). However selection of e.g. an IgG1 isotype and inclusion ofseveral mutations will potentially be able to result in immunogenicityand cause an immune response against the Fc part in humans, as such amodified Fc region not will be naturally occurring in humans.

For that reason the hzACO-1 anti-IFN-α mAb was expressed both with IgG2and IgG4 isotypes, which are both used in number of therapeuticantibodies (Salfeld J G. Biotechnol 25, 1369-1372, 2007). Unexpectedly,the expression levels of the IgG4 construct was significantly higherthan of the IgG2 hzACO-1 variant (Example 5) which will improve theproduction yield considerably. Furthermore, the IgG2 hzACO-1 moleculebut not the IgG4 variant was prone to aggregation (Example 11). This isconsidered a serious problem in drug development, as aggregation maylower yield during production, limit shelf-life and result in reducedpotency in patients due to increased immunogenicity.

Accordingly, the hzACO-1 IgG4 construct was selected for furthercharacterization in an ADCC assay in the presence of IFN-α, as well as acomplement fixation ELISA assay, as described in example 12 and 13,respectively. These data confirm that the hzACO-1 IgG4 molecule isindeed unable to cause undesired cytotoxicity. The expected lack ofpotentiation of IFN-α activity may also be confirmed in PBMCs and ECs,as described in Moll et al 2008.

In summary, the hzACO-1 IgG4 molecule appears to be a particularsuitable therapeutic molecule as it is capable of neutralizing IFN-αwithout induction of undesired sideeffects caused by Fc-mediatedeffector functions including cytotoxicity and potentiation of IFNactivity, and as it is a stable and well expressed molecule making itsuitable for manufacturing and administration to patients.

Example 15 Analysis of T-cell Epitopes

Based on standard technologies for immunogenicity predictions (De Groot,A. S, and Moise, L. Curr. Opin. Drug Discov. Devel. 10, 332-340, 2007)the pocket profile method (Sturniolo, T. et al. Nat. Biotechnol. 17,555-561, 1999) as extended by ProPred (Singh, H. & Raghava, G. P.ProPred. Bioinformatics. 17, 1236-1237, 2001) was used to predict linearT-Cell epitopes among 51 HLA-DRB alleles. The method calculates thenumber of alleles a given 9 residue long peptide within the proteinunder evaluation can bind. If a given peptide that binds many alleles isof non-human origin, this can be used as an indirect measure ofimmunogenicity. If the peptide is of human origin, no immune response isexpected due to the early negative selection of corresponding T-Cells.

It is the purpose of this example to compare the predictedimmunogenicity of a humanization procedure using the full lengthhzACO-1-kabat CDRH2 to the actual humanization procedure applied forhzACO-1.

As input sequences to the ProPred algorithm, full length ACO-1 CDR_H2with 10+10 residues added around hzACO-1-kabat CDRH2 is used:

-   APGQGLEWMG/EINPSHGRTIYNENFKS/RVTMTRDTST (CDR_H2_Full),-   together with the comparable hzACO-1 sequence-   APGQGLEWMG/EINPSHGRTIYAQKFQG/RVTMTRDTST (CDR_H2_Human)-   in the same area.

Running CDR_H2_Full through the T-Cell epitope predictor gives thefollowing 3 epitopes: WMGEINPSH (binding to 4% of HLA-DRB alleles),INPSHGRTI (6%) and FKSRVTMTR (24%). For CDR_H2_Human the first two minorepitopes are identical, whereas the last major epitope is converted toFQGRVTMTR (27%). Even though it is still a T-cell epitope, it is now afully human sequence and from the assumption, that self reactive T-Cellsare deleted by negative selection in the Thymus, this potential majorepitope in CDR_H2_Full has been removed by the CDR_H2_Human sequence.

Accordingly hzACO-1 is expected to be less immunogenic than atraditional CDR grafted humanized ACO-1.

Example 16 CDR Truncation

As described in Example 2, the mouse ACO-1 antibody was humanized by anuntraditional method. This constitutes the designing of a mask ofresidues predicted to comprise the paratope, based on a 3D model ofhzACO-1 and IFN-αA. The application of this humanization method resultedin a hzACO-1 antibody with fewer murine residues than an antibodyhumanized by simple CDR grafting, since the peptide comprising the 5C-terminal amino acids of the optimized hzACO-1 CDR H2 sequence wasidentical to the corresponding human framework sequence. In contrast,the corresponding peptide sequence in a traditionally CDR graftedhumanized antibody (the hzACO-1-kabat CDRH2) was of murine origin.Accordingly, this humanization procedure resulted in a humanizedantibody with a more human sequence, less likely to cause immunogenicityin patients. Analysis of the sequences of the CDR H2 revealed that byreducing the mouse amino acid residues in the hzACO-1, an MHC class II Tcell epitope containing the mouse amino acids is removed and replacedwith a fully human sequence, which patients would be expected to betolerant to. Accordingly, this confirmed that hzACO-1 is expected to beless immunogenic, than a traditional CDR grafted humanized ACO-1antibody.

The affinity of the hzACO-1 antibody was retained, within two-fold ofthe mouse ACO-1 antibody, as shown in Example 8. Accordingly, no furthermouse backmutations were required. Furthermore, the IFN-α subtypeprofile of the antibody, binding and neutralizing all IFN-α subtypesexcept the IFN-α1/D had been retained, as described in Examples 8, 9 and10. Despite containing fewer mouse amino acids in the CDR H2, theaffinity of the hzACO-1 was identical to the affinity and the potency ofthe hzACO-1-kabat CDRH2 antibody, containing the full length mouse CDRH2 from the mouse ACO-1 antibody (Example 8 and 9, respectively).

Furthermore, the replacement of the sequence AQK instead of NEN inposition 60-62 in heavy chain by use of the described humanizationmethod has the advantage of avoiding two asparagine that may be prone todeamidation. Deamidation changes the net charge of proteins which mayeffect stability and/or specificity. By keeping the sequence AQK thehomogeneity of hzACO-1 will be better preserved. Comparison of thehzACO-1 to the traditionally CDR grafted hzACO-1-kabat CDRH2 version,revealed that the hzACO-1 is a highly stable protein, whereas thehzACO-1-kabat CDRH2 antibody unexpectedly had a tendency to aggregate(Example 11). Aggregation is considered a serious problem in drugdevelopment, as aggregation may result in lower yield during production,limited shelf-life and reduced potency in patients due to increasedimmunogenicity. In addition, the expression levels of the hzACO-1construct was unexpectedly twice that of the hzACO-1-kabat CDRH2 variant(Example 5).

In summary, the hzACO-1 IgG4 antibody was humanized by a novel approachresulting in a therapeutic antibody with less mouse amino acids which isless likely to cause immunogenicity in patients. Despite containingfewer amino acids from the original mouse mAb, this humanized antibodyhad a comparable affinity, potency and IFN-α subtype profile as themouse antibody and a humanized version generated by traditional CDRgrafting. Furthermore, the hzACO-1 antibody is less prone to deamidationand has a higher expression level. Thus, the hzACO-1 antibody is astable and well expressed molecule suitable for manufacturing andadministration to patients.

1. A method of treating or ameliorating an IFN-α related condition ordisease, said method comprising administering to a subject in needthereof a [prophylactically or] therapeutically effective amount of anantibody that binds human interferon-α and comprises a VH domaincomprising SEQ ID NO:3 and a VL domain comprising SEQ ID NO:6.
 2. Themethod of claim 1, wherein said IFN-α related condition or disease isSLE.
 3. The method of claim 1, wherein said IFN-α related condition ordisease is psoriasis.
 4. A method of treating or ameliorating an IFN-αrelated condition or disease, said method comprising administering to asubject in need thereof a [prophylactically or] therapeuticallyeffective amount of an antibody comprising at least one light chain thatcomprises the CDRs: V_(L)1 having the amino acid sequence of SEQ IDNO:18; V_(L)2 having the amino acid sequence of SEQ ID NO:19; and V_(L)3having the amino acid sequence of SEQ ID NO:20; and at least one heavychain that comprises the CDRs: V_(H)1 having the amino acid sequence ofSEQ ID NO:15; V_(H)2 having the amino acid sequence of SEQ ID NO:21; andV_(H)3 having the amino acid sequence of SEQ ID NO:17.
 5. The method ofclaim 4, wherein said IFN-α related condition or disease is SLE.
 6. Themethod of claim 4, wherein said IFN-α related condition or disease ispsoriasis.
 7. The method of claim 1, wherein said IFN-α relatedcondition or disease is type I diabetes.
 8. The method of claim 4,wherein said IFN-α related condition or disease is type I diabetes. 9.The method of claim 1, wherein said antibody is an IgG4 isotypeantibody.
 10. The method of claim 4, wherein said antibody is an IgG4isotype antibody.